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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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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Biology

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

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 ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...