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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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-associated+protein+kinase+inhibition+enhances+airway+epithelial+Basal-cell+proliferation+and+lentivirus+transduction.">Rho-associated protein kinase inhibition enhances airway epithelial Basal-cell proliferation and lentivirus transduction.</a> Am J Respir Cell Mol Biol. 49, 341-347 (2013).</li><li>Manzanares, D., 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=Functional+apical+large+conductance,+Ca2+-activated,+and+voltage-dependent+K++channels+are+required+for+maintenance+of+airway+surface+liquid+volume.">Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume.</a> J Biol Chem. 286, 19830-19839 (2011).</li><li>Schmid, A., 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=Real-time+analysis+of+cAMP-mediated+regulation+of+ciliary+motility+in+single+primary+human+airway+epithelial+cells.">Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells.</a> J Cell Sci. 119, 4176-4186 (2006).</li><li>Unwalla, H. J., Ivonnet, P., Dennis, J. S., Conner, G. E., Salathe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta1+and+cigarette+smoke+inhibit+the+ability+of+beta2-agonists+to+enhance+epithelial+permeability.">Transforming growth factor-beta1 and cigarette smoke inhibit the ability of beta2-agonists to enhance epithelial permeability.</a> Am J Respir Cell Mol Biol. 52, 65-74 (2015).</li><li>Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z., Gijsbers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+lentiviral+vector+titration+methods.">Comparison of lentiviral vector titration methods.</a> BMC Biotechnol. 6, (2006).</li><li>Schneiderman, S., Farber, J. L., Baserga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+decreasing+the+toxicity+of+polyethylene+glycol+in+mammalian+cell+hybridization.">A simple method for decreasing the toxicity of polyethylene glycol in mammalian cell hybridization.</a> Somatic Cell Genet. 5, 263-269 (1979).</li><li>Schmid, A., 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=Soluble+adenylyl+cyclase+is+localized+to+cilia+and+contributes+to+ciliary+beat+frequency+regulation+via+production+of+cAMP.">Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP.</a> J Gen Physiol. 130, 99-109 (2007).</li><li>Manzanares, D., 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=Airway+Surface+Dehydration+by+Transforming+Growth+Factor+beta+(TGF-beta)+in+Cystic+Fibrosis+Is+Due+to+Decreased+Function+of+a+Voltage-dependent+Potassium+Channel+and+Can+Be+Rescued+by+the+Drug+Pirfenidone.">Airway Surface Dehydration by Transforming Growth Factor beta (TGF-beta) in Cystic Fibrosis Is Due to Decreased Function of a Voltage-dependent Potassium Channel and Can Be Rescued by the Drug Pirfenidone.</a> J Biol Chem. 290, 25710-25716 (2015).</li><li>Manzanares, D., 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=IFN-gamma-mediated+reduction+of+large-conductance,+Ca2+-activated,+voltage-dependent+K++(BK)+channel+activity+in+airway+epithelial+cells+leads+to+mucociliary+dysfunction.">IFN-gamma-mediated reduction of large-conductance, Ca2+-activated, voltage-dependent K+ (BK) channel activity in airway epithelial cells leads to mucociliary dysfunction.</a> Am J Physiol Lung Cell Mol Physiol. 306, L453-L462 (2014).</li><li>Chen, X., 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=A+soluble+adenylyl+cyclase+form+targets+to+axonemes+and+rescues+beat+regulation+in+soluble+adenylyl+cyclase+knockout+mice.">A soluble adenylyl cyclase form targets to axonemes and rescues beat regulation in soluble adenylyl cyclase knockout mice.</a> Am J Respir Cell Mol Biol. 51, 750-760 (2014).</li><li>Ransford, G. 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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 ...

The overall goal of this methodology is to achieve highly efficient transduction of primary human bronchial epithelial cells using lentiviruses. This method can help researchers who want to use primary human bronchial epithelial cells rather than cell lines, and might be adaptable to other difficult to transduce cell types. The main advantage of this technique is a high transduction efficiency maintained throughout differentiation into a psuedostratified epithelium.The protocol should be executed in line with institutional and governmental regulations under enhanced BSL-2 conditions in the USA. After preparing HEK medium in a Collagen I coated dish, according to the text protocol, use 10 milliliters of the medium to plate one million HEK cells onto the coated dish. Incubate the dish at 37 degrees Celsius, and 5%carbon dioxide, until the cells are 50 to 60%confluent.This will be considered Day 0. On Day 0, in a laminar flow cabinet, using a commercial transfection kit, bring reagents to room temperature. For each 10 centimeter dish of HEK293 cells, in a 15 milliliter centrifuge tube, combine the following:4.5 micrograms of pMD2-VSVG envelope DNA, 7.5 micrograms of pMDLg/pRRE packaging DNA, 3.75 micrograms of pRSV-rev DNA, 10 micrograms of lentiviral expression vector, and 86 microliters of a 2 Molar calcium solution.Then add sterile water to a final volume of 700 microliters. Still in a laminar flow cabinet, while vortexing, add 700 microliters of 2x Hepes Buffered Saline dropwise, and incubate at room temperature for 30 minutes. Early in the incubation, deposit 5 microliters of the mixture on a small dish.Under a microscope, with a 10x objective, check the drop by slowly focusing up and down. A fine precipitate should be visible, confirming the calcium phosphate precipitation process. The most critical step is the precipitate.If none is present, it may indicate a bad plasmid preparation or miscalculation of plasmid or transfection reagents. Proceeding to the next step with our precipitate, may result in low virus preparation and/or pro-transductions. Stop the precipitation after 30 minutes by adding 10 milliliters of HEK medium and pipetting up and down to mix.Then, remove the medium from the dish of HEK cells and carefully and slowly add the precipitate solution along the edge of the dish. The next day, remove and discard the medium containing the precipitate and replace it with 10 milliliters of HEK medium. Under the microscope, check for fine precipitate.It should be visible in the dish in empty spaces, between the cells. The following day, using a syringe, collect the medium, and filter it through a 0.45 micrometer filter, into a 15 milliliter centrifuge tube containing 3.8 milliliters of 40%PEG solution. Invert the tube five times to mix, and store at four degrees Celsius for at least 24 hours, until all the virus collections are complete.On Days 3 and 4, repeat the medium collection and filtering. Then use 10%bleach to decontaminate the dish before discarding. Centrifuge all of the collection tubes at 1, 650 x g and four degrees Celsius for 20 minutes to pellet the virus.Remove and discard the supernatant, and spin again for five minutes to collect and remove any remaining PEG supernatant. Use 200 microliters of bronchial epithelial growth medium, or BEGM, without amphotericin B, to re-suspend each pellet. Then, pull them together, and prepare 200 microliter aliquots.Store the aliquots at 80 degrees Celsius. In a Collagen I coated dish, plate one million HBE cells in 10 milliliters of BEGM medium. Incubate at 37 degrees Celsius, and 5%carbon dioxide, replacing the medium every day for a total of four days.After coating permeable support inserts with Collagen IV, according to the text protocol, to transduce 80 to 90%confluent HBE cells, remove the medium, and add three milliliters of 0.1%trypsin, and one milliMolar EDTA PBS. Incubate at 37 degrees Celsius for five minutes. When the cells have detached, add three milliliters of Soybean Trypsin Inhibitor, suspend the cells, and transfer to a 15 milliliter centrifuge tube.Then pellet the cells by spinning at 460 x g and room temperature for five minutes. Remove the supernatant, and use three milliliters of BEGM to re-suspend the pellet and proceed with counting. Then, after counting the cells, add more BEGM so that each 12 millimeter permeable support insert contains 200, 000 cells and 400 microliters.Using a multiplicity of infection, or MOI, factor of four, add the appropriate volume of virus to the cell suspension. Then, add hexidine methylene bromide, at a final concentration of two micrograms per milliliter. Dispense 400 microliters of the cell mixture per 12 millimeter permeable support insert into the apical compartment, and add one milliliter of BEGM alone to the basolateral compartment.Incubate overnight at 37 degrees Celsius and 5%carbon dioxide. The following day, remove the apical and basolateral medium, and use PBS to wash the cells. Then, use air-liquid interface, or ALI, medium to replace both compartments.Finally, when the cells reach confluency, remove the apical fluid, and continue to change the medium in the bottom compartment every other day, until the cells reach maturity. Refer to the text protocol for additional details. Shown here are HEK cells at approximately 50 to 60%confluency, at which point, the cells are optimal for transfection.This figure illustrates the precipitate in a drop in the transfection mixture. A precipitate that looks more like sand grains, rather than agglomerates, will increase the efficiency of the transfection. This image shows an example of primary HBE cells, that are approximately 80%confluent, prior to their trypsinization and transduction.In this EGFP transduction experiment, using an MOI of four on permeable support inserts, a nearly 100%transduction rate was achieved. Once mastered, the transfection portion of this protocol can be done in 45 minutes. The entire protocol, however, will take at least 10 days.While attempting this procedure, it is important to remember that this protocol should be executed in line with institutional and governmental regulations on the BSL-2+conditions. Following this procedure, primary human bronchial epithelial cells, rather than cell lines, can easily be manipulated to over-express or silence proteins of specific genes. These primary cells are more representative of in vivo tissues.After watching this video, you should have a good understanding of how to achieve highly efficient transductions of primary human bronchial epithelial cells using lentiviruses.

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18.9K ビュー.  University of Miami, Miller School of Medicine. The overall goal of this methodology is to achieve highly efficient transduction of primary human bronchial epithelial cells using lentiviruses. This method can help researchers who want to use primary human bronchial epithelial cells rather than cell lines, and might be adaptable to other difficult to transduce cell types. The main advantage of this technique is a high transduction efficiency maintained throughout differentiation into a psuedostratified epithelium.The protocol should ...