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 min.</li>
	<li>Plate HEK cells onto the coated dish at a density of 1 x 106 cells in 10 ml HEK media and incubate at 37 &#176;C and 5% CO2.</li>
	<li>When HEK cells are 50-60% confluent (Figure 1a, 2-3 days after plating), proceed to stage 2. Visual assessment of confluency is sufficient. No quantification is needed. 
	Note: HEK cells are cultured in HEK media (see above). When they reach confluency, they can be split (e.g. 1/5) or frozen down for later use.</li>
</ol>
2. Stage 2: Production and Concentration of Lentiviruses (LVs) in HEK Cells
Note: The protocols should be executed in line with institutional and governmental regulations under BSL-2+ (enhanced BSL-2) conditions in the USA.
<ol>
	<li>Transfect using a calcium precipitation procedure (lipofectamine and other transfection agents were not as efficient as the calcium precipitation procedure). Use of a commercial transfection kit is recommended to assure reproducibility, though the procedure is modified as follows. Bring all reagents to room temperature. This is day 0.</li>
	<li>For each 10 cm dish of HEK293 cells, mix 4.5 &#181;g of envelope DNA pMD2-VSVG, 7.5 &#181;g packaging DNA pMDLg/pRRE, 3.75 &#181;g DNA pRSV-rev, 10 &#181;g DNA of lentiviral expression vector, 86 &#181;l of a 2 M calcium solution, and sterile water in a 15 ml centrifuge tube to a final volume of 700 &#181;l (the latter two solutions are provided in the commercial transfection kit).</li>
	<li>Drop-wise, add 700 &#181;l of 2x HEPES-buffered saline (HBS, provided in the transfection kit) while vortexing (in the laminar flow cabinet) and incubate at room temperature for 30 min (perform step 4 while waiting).</li>
	<li>During the early incubation time, deposit 5 &#181;l of the mix on a slide. Check the droplet under a microscope with a 10X objective, focusing slowly up and down. A fine precipitate should be visible, confirming the calcium phosphate precipitation process (Figure 1b).</li>
	<li>Stop the precipitation after 30 min by adding 10 ml of HEK cells media and pipette up and down to mix.</li>
	<li>Take the dish of HEK cells from step 2.1-2.5, and remove the medium (day 0). Add the precipitate solution from step 2.5 carefully and slowly along the edge of the dish.</li>
	<li>On the next day (day 1), remove and discard the medium containing the precipitate and replace it with 10 ml of HEK medium. Check for the fine precipitate under the microscope: it should be visible in the dish in empty spaces between the cells (Figures 1c and d).</li>
	<li>On day 2, collect media with a syringe, filter through a 0.45 &#181;m syringe filter into a 15 ml centrifuge tube containing 3.8 ml of a 40% polyethylene glycol (PEG) solution. Invert 5x to mix and store at 4 &#176;C for at least 24 hr (for the PEG precipitate to form, but no longer than 5 days) until all of the virus collections are complete.</li>
	<li>Repeat step 2.8 on days 3 and 4. On day 4 do not replace with HEK medium but decontaminate with 10% bleach and discard the dish.</li>
	<li>Centrifuge all the collection tubes (usually all collections together on day 5) at 1,650 x g for 20 min at 4 &#176;C to pellet the virus. Remove and discard the supernatant, and spin again at 1,650 x g for 5 min at 4 &#176;C to collect and remove any remaining of PEG supernatant.</li>
	<li>Resuspend the pellet of each tube in 200 &#181;l of bronchial epithelial growth medium (BEGM) without amphotericin B 8. Pool them together and aliquot at 200 &#181;l. Store virus at -80 &#176;C. Aliquot an additional 10 &#181;l to perform the HIV-1 Gag (p24) antigen ELISA assay.</li>
	<li>Using a commercial ELISA kit, perform a p24 antigen assay according to manufacturer&#39;s protocol. The assay gives an estimate of viral p24 in pg/ml.</li>
</ol>
3. Stage 3: Culture Primary HBE Cells
<ol>
	<li>Plate HBE cells in 10 ml BEGM media (with 100 &#181;l amphotericin B if passage 0 cells are used to eliminate possible fungal contamination) at a density of 1 x 106 cells in a collagen I coated 10 cm tissue culture dish. Incubate at 37 &#176;C and 5% CO2.</li>
	<li>On the next day, remove the medium and replace with 10 ml BEGM (with 100 &#181;l amphotericin B for passage 0 cells) for a total of 4 days. Return to incubator.</li>
	<li>After 4 days, use only BEGM without amphotericin B. Change media every other day.</li>
	<li>When cells are 80-90% confluent (Figure 2a; ~7 days after plating), proceed with transduction (pay attention to 3.5 for preparation before transduction). Again, visual assessment of confluency is sufficient. No quantification is needed.</li>
	<li>Prior to transduction, coat the permeable support inserts with a collagen IV solution diluted 1/10 with sterile distilled water. Add 200 &#181;l to each insert and let dry overnight in the laminar flow cabinet.</li>
</ol>
4. Stage 4: Transduction of HBE Cells
<ol>
	<li>Remove media, add 3 ml of 0.1% trypsin in 1 mM ethylenediaminetetraacetic acid in phosphate buffered saline (EDTA/PBS) and incubate 5 min at 37 &#176;C and 5% CO2. Check under the microscope (10X objective) to see if all the cells are detached. If not, return to the incubator for an additional 5 min.</li>
	<li>When all cells are detached, add 3 ml of soybean trypsin inhibitor (SBTI 1 mg/ml), mix and transfer to a 15 ml centrifuge tube. Pellet the cells by spinning at 460 x g for 5 min at room temperature.</li>
	<li>Remove supernatant and resuspend pellet into 3 ml of BEGM and proceed with counting.</li>
	<li>Resuspend HBE cells (now passage 1 or P1) at a ratio of 2 x 105 cells per one 12 mm permeable support insert in 400 &#181;l of BEGM (see Table 2). Extrapolate these numbers based on the amount of permeable support inserts that will be transduced.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Surface</td>
			<td>Surface area</td>
			<td>Cell count</td>
			<td>Media</td>
			<td>Volume (media)</td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>52.7 cm2</td>
			<td>1 x 106</td>
			<td>BEGM</td>
			<td>10 ml</td>
		</tr>
		<tr>
			<td>12 mm filter</td>
			<td>1.13 cm2</td>
			<td>2 x 105</td>
			<td>BEGM</td>
			<td>400 &#956;l</td>
		</tr>
		<tr>
			<td>24 mm filter</td>
			<td>4.52 cm2</td>
			<td>8 x 105</td>
			<td>BEGM</td>
			<td>800 &#956;l</td>
		</tr>
	</tbody>
</table>
Table 2: Media extrapolation per cell count.
<ol start="5">
	<li>Using a multiplicity of infection (MOI) factor of 4, add the appropriate volume of virus to the cell suspension.</li>
	<li>Add hexadimethrine bromide to a 2 &#181;g/ml final concentration.</li>
	<li>Dispense 400 &#181;l of the mix (BEGM, HBE cells, virus and hexadimethrine bromide) per permeable support insert into the apical compartment, and add 1 ml of BEGM alone to the basolateral compartment. Incubate overnight at 37 &#176;C and 5% CO2. Always plate HBE cells without virus as control and test puromycin selection if applicable.</li>
	<li>On the next day, remove apical and basolateral media, wash with PBS, and replace both compartments with air-liquid interface (ALI) media 8.</li>
	<li>When cells are confluent, remove apical fluid (known as establishing an air-liquid interface). Continue changing the medium (ALI) in the bottom compartment every other day until they reach full maturity; usually 3 to 4 weeks later. 
	Note: If the lentiviral vector contains a selection gene such as puromycin, cells can be selected by adding puromycin according to a selection concentration curve. For HBE cells, a concentration of 1 &#181;g/ml has been determined to be ideal for consistent selection of transduced cells. However, this concentration might differ for other cells or conditions and should be determined by killing curves. Addition of puromycin can start as early as one day after the media has been replaced by ALI media or later (2-3 days) to allow the full expression of the selection gene. Be sure to add puromycin to one insert that has not been transduced. Puromycin can be added until the cells are fully differentiated.</li>
</ol>

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The authors have nothing to disclose.

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 reagents, a pH issue, or a bad plasmid preparation. If the precipitate is missing, there is a high likelihood that none will be visible the next day. Furthermore, adding the transfection mix or HEK media too fast may cause the HEK cells to detach, hindering transfection and yield. Another indication of a good yield is at day 6 before the centrifugation step: a white precipitate should be visible in the bottom of the tube. A bad transfection with almost no virus will not create this white precipitate. During the transduction process, it is crucial to use primary HBE cells that have been de-differentiated and that are in a stem-cell like state in order to achieve high transduction efficiencies. The use of a vector containing a fluorescent protein such as eGFP or mCherry that is easily visualized can be a good tool.
Modifications could be made to adapt this protocol to different primary cells. One approach would be to reduce the calcium concentration in the media. As seen in Figure 3a, using BEGM media that contains a lower concentration of calcium resulted in significant higher transduction efficiency in primary HBE cells. Another approach could be to increase the MOI. As shown in Figure 3d, the higher the MOI, the better the transduction efficiency. However, using a higher MOI might become toxic to some cells.
Nevertheless, there are some limitations to this protocol. This technique has been developed using passage 1 HBE cells. Lungs rejected for transplant were obtained with appropriate consent for research conforming to the standards set by the declaration of Helsinki. Obtaining primary HBE cells from commercial sources can be expensive. These cells are usually also only available at a higher passage. This protocol has not been tested on passages higher than 2. Another limiting factor is the p24 ELISA antigen assay. Several companies offer the kit but at high cost. In addition, the viral concentration given by the assay, is only an approximate value since the test detects lentivirus p24 and free p24 released during transient transfection 17. Therefore, this evaluation of MOI is only an estimate. Finally, using different expression constructs (larger or smaller in size) may not give the same results. For instance, different efficiencies were recorded using mCherry and eGFP (data not shown).
There are advantages to this protocol over other methods. One of them is the transfection process. In fact, the proposed protocol can yield such high virus titers that the collected media containing viruses will no longer need to be concentrated and could be used directly to transduce cells. Polyethylene glycol (PEG), used in this protocol to concentrate virus, has been known to be toxic to mammalian cells 18. In addition, if higher MOI were used, this would increase the toxicity risk even more. However, if the PEG concentration step becomes obsolete, researchers that have been unsuccessful to transduce due to toxicity effects might reconsider the use of lentiviruses. Another advantage of this protocol is the lack of differences in transduction efficiencies on plastic or permeable support inserts. The benefit of being able to transduce on plastic is that fewer cells and virus particles are needed. As a result, the cells can be expanded post transduction and transferred onto permeable support inserts when confluent (data not shown).
The choice of vectors depends on several issues, such as selection of transduced cells with puromycin or expression of marker proteins such as green fluorescent protein (GFP). There are multiple commercial vectors available that are suitable (pRRLsinPPT.CMV.MCS.Wpre, pCDH-EF1-MCS-IRES-Puro, pCDH-EF1-MCS-T2A-Puro, pGEM-T Easy) 15,19-21. Selection of promoters poses challenges, since the commonly used CMV promoter is silenced in differentiated airway epithelial cells 15. For ciliated cell-specific expression, use of the fox-j1 promoter is recommended. Alternatively, the EF-1 promoter can be used for expression in all airway cells 20,21. If protein overexpression is sought, the size of the insert will determine, in part, transduction efficiency. However, large sizes can be accommodated as seen by successful transduction of full-length soluble adenylyl cyclase 22.The lab has published several successful overexpression constructs 15,16,20-22. For shRNA expression (usually under the U6 or H1 promoter), multiple constructs will have to be explored. This can be tedious with tests done at the end of differentiation (PCR and western blotting), but the process cannot be abbreviated since non-differentiated cells may not be a good representation of success for cells undergoing differentiation. Again, the lab has published multiple successful shRNA constructs 14,20,21,23.
Using this protocol, more researchers might be enabled to significantly improve previous poor transductions. Some might find it useful to increase the efficiencies by making a few adjustments. Some might use this tool to discover new biological processes or to be able to prove concepts, both using knockdown or overexpression strategies.

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><tbody><tr><td>HEK293T/17 cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</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 2 M Calcium solution, 2x HEPES-Buffered solution (HBS) and sterile H&lt;sub&gt;2&lt;/sub&gt;O</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 12 mm</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 2 mg/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 grains rather than agglomerates, the more efficient the transfection will be. In this picture, the precipitate is a bit coarse. The precipitate is a reliable indicator whether the transfection will produce a high-titer of virus. If any of the ingredients is either missing or of poor quality (vector DNA for instance), agglomerates will form, which is an ominous sign. If this is the case, it is wiser to abort and restart the procedure, making sure that all the ingredients are in the mix, and/or checking the quality of the vector DNA and packaging DNA. Figure 1c shows the same dish as A, after overnight incubation. The precipitate is (and should be) visible in between cells. We noticed that the cells do not multiply much during the 24 hr following transfection.
Figure 2 shows how HBE cells look before and 3 days after transduction. Figure 2a depicts approximately 80% confluent primary HBE cells prior to trypsinization and transduction. A 10 cm dish at 80% confluence yields approximately 4 x 106 cells. Figure 2b shows nearly 100% transduction of undifferentiated P1 HBE cells using an MOI of 4 on permeable support insert. When HBE cells are plated on these permeable support inserts, the cells are relatively flat. On this picture, the height of the cells is only a few microns which explains why the 0.4 &#181;m pores are visible though the cells.
Figure 3 shows optimization experiments leading to the presented protocol. All experiments have been performed in triplicates on collagen I coated wells (24-well plate), with 2 x 105 primary HBE cells per well. Most experiments used an MOI of 0.4 with a 2 &#181;g/ml final concentration of hexadimethrine bromidein BEGM media. Fluorescent cells were counted 3 days after transduction. Even though the initial optimization of the protocol has been performed on plastic, the results have been confirmed on permeable support inserts. In addition, attempts to transduce fully differentiated P1 HBE cells did not succeed (data not shown), confirming previous reports. As seen in Figure 3a, a higher percentage of HBE cells are transduced in BEGM media compared to ALI. If the cells are plated the day before transduction in BEGM media, the efficiency is significantly decreased compared to transducing while cells are attaching (Figure 3b). When P1 HBE cells are plated the day before transduction in ALI media (instead of BEGM), the transduction efficiency decreases even further (data not shown). The difference between these 2 media is the concentration of calcium. BEGM media contains a low concentration of calcium (0.1 mM) compared to ALI media (1 mM). Calcium forms and maintains tight junctions and therefore, could make it difficult for the virus particles to cross 9 and access their receptors at the basolateral membrane. In fact, some infections were successful at low levels by disrupting tight junctions using ethylene glycol tetraacetic acid (EGTA) for instance 10. As stated previously, fully differentiated P1 HBE cells are refractory and can only be transduced with low efficiencies 5-7, which could be due to tight junctions in high calcium containing media. Another explanation for low transduction efficiency in fully differentiated cells is an active mucociliary transport mechanism where mucus could act as a barrier. Or, as reported by Bals et al., the stage of differentiation in which HBE cells are, is a determining factor of transduction efficiency 10. The fact that more cells get transduced in BEGM than in ALI media supports this statement (Figure 3a).
As seen in Figure 3b, an increase of almost 50% transduction efficiency was observed when the cells were transduced while attaching compared to cells plated the day prior. In their study, Ricks et al. showed an increase of 20% when transduction was performed after trypsinization 11. Together, these results suggest that media and the state of maturity of cells is key to transduction success.
The panel in Figure 3c, demonstrates hexadimethrine bromideplaying atrivial role in transduction efficiency. Nevertheless, its use is suggested in this protocol since it has no negative impact but could potentially be beneficial. Hexadimethrine bromide is a cationic polymer that is used in virology to enhance transduction efficiencies 12. Others have published the use of protamine, another cationic polymer, but no significant difference was found regarding transduction efficiency compared to hexadimethrine bromide 13. Hexadimethrine bromide does not impact growth nor differentiation 14-16.
Finally, different MOIs were investigated in Figure 3d: the highest transduction efficiency occurred with an MOI of 4. There was no significant difference between MOI 0.4 and 4, however, MOI 4 transduced 100% of HBE cells whereas MOI 0.4 only transduced 75%. The MOI defines how many virus particles will have the potential to infect one cell. There are different methods to determine the MOI. One of them is to define the number of transduction units (TU). Using an ELISA, the amount of p24 antigen can be determined in pg/ml. Viral p24 in picograms can be converted in TU, which are necessary to calculate the MOI. The MOI is calculated by dividing the number of TU by the number of cells. There are 10-100 transduction units (TU) per picogram p24. All references in the text regarding MOI are based on calculations with 100 TU per picogram p24. (http://tronolab.epfl.ch/webdav/site/tronolab/shared/protocols/TUvsp24.html).
<img alt="Figure 1" src="/files/ftp_upload/54176/54176fig1.jpg" /> 
Figure 1: Transfection of HEK 293T cells. (a) HEK 293T cells grown to 50-60% confluency as seen under a 10X objective. (b) Precipitate visible in a 5 &#181;l drop 5 min after mixing all reagents under the microscope (10X objective). (c) Precipitate in the dish next to the cells,following overnight transfection (10X objective). (d) Magnification of center of (c). White arrow pointing to precipitate. Scale bar = 100 &#181;m; is the same for a, b, and c. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54176/54176fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54176/54176fig2.jpg" /> 
Figure 2: Transduction of NHBE cells. (a) HBE cells grown in a collagen I coated dish to 80-90% confluency (approximately 4 x 106 cells) in BEGM media for 5 days (10X objective). Scale bar = 100 &#181;m. (b) eGFP transduced HBE cells on permeable support inserts (12 mm), 3 days post transduction (confocal, 20X). At this state, HBE cells are flat and the membrane&#39;s pores are visible. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54176/54176fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54176/54176fig3.jpg" /> 
Figure 3: Optimization of transduction protocol. (a) Impact of media on transduction efficiency. (b) Comparison of transduction in HBE cells plated the day before and transduction of cells while attaching. (c) Influence of hexadimethrine bromide on transduction efficiency. (d) Evaluation of improved transduction efficiencies using different MOIs. Analysis was performed using Student&#39;s t-test or Kruskal-Wallis and Dunn&#39;s multiple comparison test. Error bars represent SE and all * represent p &#60;0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54176/54176fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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19.2K Views.  University of Miami, Miller School of Medicine. 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.

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

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Immunology and Infection

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Developmental Biology

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

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

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