We appreciate support from Mizuno Takako for IFC and H and E staining, Vanessa Garcia for tissue sectioning&#160; and&#160; Anika S Chandrasekaran for helping with manuscript preparation. This work is supported by National Institutes of Health (5RO1HL135163-04, PO1HL108793-08) and Celgene IDEAL Consortium.

Human lung tissue was obtained from deceased tissue donors in compliance with consent procedures developed by International Institute for the Advancement of Medicine (IIAM) and approved by the Cedars-Sinai Medical Center Internal Review Board.
1. Tissue processing for isolation of lung cells from either tracheo-bronchial or small airway/parenchymal (small airways and alveoli) regions
<ol>
	<li>Prepare and autoclave all dissection instruments, glassware and the appropriate solutions one day prior to cell isolation.</li>
	<li>Upon receiving lung tissue, identify and separate the proximal and distal regions. The trachea and bronchi are considered &#34;proximal&#34;. For the purposes of this protocol,&#160;trachea and the first 2-3 generations of bronchi are disected and used for the isolation of &#8220;proximal&#8221; airway epithelium. Small airways of 2 mm or less in diameter and surrounding parenchymal tissue are, for the purpose of this protocol, considered as &#34;distal&#34;&#160;lung epithelium (Figure 1A). 
	NOTE: All procedures involving processing of human lung tissue should be performed in a biosafety cabinet with use of appropriate personal protective equipment.</li>
</ol>
2. Enrichment and subsetting of small airway and alveolar epithelial progenitor cells from distal lung tissue
<ol>
	<li>Distal tissue preparation
	<ol>
		<li>Place the distal lung tissue in a sterile Petri dish (150 x 15 mm). Dice tissue into approximately 1 cm3 pieces and place in a clean 50 mL tube.</li>
		<li>Wash the tissue 3x with chilled HBSS, discarding HBSS wash each time to remove blood and epithelial lining fluid.</li>
		<li>Place the tissue in a new Petri dish and blot dry with sterile anti-lint wipes. Using forceps and scissors, remove as much visceral pleura (a delicate transparent membrane that covers surface of the lung) as possible.</li>
		<li>Use scissors to mince tissue into pieces of approximately 2 mm diameter. Transfer minced tissue into a clean Petri dish and mince further by chopping it to an approximate size of 1 mm with a sterile single sided razor blade.</li>
	</ol>
	</li>
	<li>Enzyme digestion 
	NOTE: Liberase stock solution is 5 mg/mL (100x) and DNase stock is 2.5 mg/mL (100x) (Table of Materials).
	<ol>
		<li>Add 50 &#181;g/mL Liberase and 25 &#181;g/mL DNase into sterile HBSS in a 50 mL conical tube.</li>
		<li>Transfer approximately 2-3 g of minced tissue to a new 50 mL conical tube with 25 mL of HBSS, containing Liberase and DNase. Incubate for 40-60 min at 37 &#176;C with continuous shaking using a mixer set at 900 rpm. After 30 min of incubation, triturate digested tissue using a 30 mL syringe without a needle to avoid formation of clumps and continue with the incubation. 
		NOTE: Incubation time with the enzymes can vary depending on the type or condition of the tissue. For example, enzymatic digestion of normal tissue takes approximately 45 min. However, fibrotic tissue from idiopathic pulmonary fibrosis samples can require a longer incubation time of up to 60 min. Therefore, monitor tissue carefully during this step to prevent damage to the surface markers, which is crucial for FACS.</li>
	</ol>
	</li>
	<li>Single cell isolation
	<ol>
		<li>Triturate the tissue by drawing 5x through a 16 G needle fitted to a 30 mL syringe. Draw the tissue suspension into a wide-bore pipette and pass through a series of cell strainers (500 &#956;m, 300 &#956;m, 100 &#956;m, 70 &#956;m, 40 &#956;m) under vacuum pressure. Wash the strainer with 20 mL of HBSS+ buffer to collect remaining cells.&#160;The recipe for HBSS+ buffer can be found in Table of Materials.</li>
		<li>Add an equal volume of HBSS+ buffer, after 45 min to the filtrate to inhibit Liberase activity and prevent over-digestion.</li>
		<li>Centrifuge filtrates at 500 x g for 5 min at 4 &#176;C. Carefully remove and discard the supernatant. Add 1 mL of Red Blood Cell (RBC) lysis buffer to the pellet, gently rock the tube to dislodge the pellet and incubate on ice for 1 min. 
		NOTE: The amount and time in the RBC lysis solution depends on the size of the pellet. It is important to maintain the cells on ice and monitor time in RBC lysis solution carefully to prevent lysis of target cells. If RBC lysis is insufficient, repeat the step.</li>
		<li>Add 10-20 mL of HBSS+ buffer to neutralize RBC lysis buffer. Centrifuge filtrates at 500 x g for 5 min at 4 &#176;C.</li>
		<li>If lysed red blood cells (ghost cells) form&#160;a cloudy layer above the cell pellet, resuspend pellet in 10 mL of HBSS+ buffer and strain the suspension through 70 &#956;m cell strainer to eliminate the ghost cells. Centrifuge the filtrate at 500 x g for 5 min at 4 &#176;C and proceed with further steps.</li>
	</ol>
	</li>
	<li>Depletion of immune cells and endothelial cell (optional step)
	<ol>
		<li>Deplete CD31+ endothelial cells and CD45+ immune cells from the pool of total cells using the CD31 &#38; CD45 microbeads conjugated to monoclonal anti-human CD31 and CD45 antibody (isotype mouse IgG1) and LS columns in accordance to the manufacturer&#8217;s protocol (Table of Materials).</li>
		<li>Collect the flow through, consisting primarily of epithelial and stromal cells, in a fresh sterile tube and centrifuge it at 500 x g for 5 min at 4 &#176;C. Perform a cell count to ascertain the total number of cells in the flow through.</li>
	</ol>
	</li>
	<li>Cell surface staining for fluorescence associated cell sorting (FACS)
	<ol>
		<li>Resuspend 1 x 107 cells per 1 mL of HBSS+ buffer. Add primary antibodies at the required concentration and incubate the cells for 30 min at 4 &#176;C in the dark. In this study, fluorophore conjugated primary antibodies were used unless otherwise stated. Details of antibody sources and titers are described in Table of Materials. 
		NOTE: HTII-280 is currently the best surface reactive antibody that allows subsetting of distal lung cells into predominantly airway (HTII-280-) and alveolar type 2 (HTII-280+) cell fractions. A caveat to this strategy is that AT1 cells are not stained using this method and are poorly represented due to their fragility. However, AT1 cells are poorly represented in distal lung preps, presumably due to their fragility and loss during selection of viable cells by FACS and thus only represent a rare contaminant of the airway cell fraction.</li>
		<li>Wash cells by adding 3 mL of HBSS+ buffer and centrifuge at 500 x g for 5 min at 4 &#176;C.</li>
		<li>If using unconjugated primary antibodies, add required concentration of an appropriate fluorophore conjugated secondary antibody and incubate for 30 min on ice. Wash off excess secondary antibody by adding 3 mL of HBSS+ buffer and centrifuge at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend cells in HBSS+ buffer per 1 x 107 cells/ mL. Filter cells into 5 mL polystyrene tubes through a strainer cap to ensure formation of a single cell suspension. Add DAPI (1 &#956;g/mL) to stain permeable (dead) cells. 
		NOTE: It is essential to use appropriate single-color and fluorescence minus one (FMO) controls (i.e., antibody staining cocktail minus one antibody each), to minimize false positives during FACS. In this study, positive and negative selection beads were used for empirical compensation for overlap of emission spectra between fluorophores (Table of Materials). FACS enrich cell types of interest. Viable epithelial cells are enriched based upon their CD45-negative, CD31-negative, CD236-positive cell surface phenotype and negative staining for DAPI. This epithelial cell fraction can be further subsetted based on staining for cell type-specific surface markers, such as specific staining for HTII-280-positive cells that are enriched for AT2 cells. In contrast, negative selection for HTII-280 allows the enrichment of small airway epithelial cells such as club and ciliated cells (Figure 2).</li>
	</ol>
	</li>
</ol>
3. Enrichment and subsetting of epithelial progenitor cells from tracheo-bronchial airways
<ol>
	<li>Tissue preparation
	<ol>
		<li>Dissect out proximal airways (trachea/bronchi) from the lungs. Open airways along their length using scissors to expose the lumen and add 50 &#181;g/mL Liberase to fully cover the tissue.</li>
		<li>Incubate for 20 min at 37 &#176;C with continuous shaking using a thermo mixer set at 900 rpm.</li>
		<li>Remove the proximal airway from the centrifuge tube and place it in a sterile Petri dish (150 x 15 mm). Gently scrape the surface of the airway using a scalpel to completely strip luminal epithelial cells from the tissue.</li>
		<li>Wash the Petri dish with 5 mL of sterile HBSS+ buffer to collect all dislodged luminal epithelial cells and transfer the dislodged cells to 50 mL conical centrifuge tube. Triturate the suspension by drawing 5x through 16 G needle and 18 G needle fitted to a 10 mL syringe to get single cell suspension.&#160;</li>
		<li>Centrifuge the suspension at 500 x g for 5 min at 4 &#176;C. Resuspend the pellet in fresh HBSS+ buffer and store these luminal airway cells on ice, ready to be combined with the single cell suspension generated from the minced proximal airways in the upcoming steps.</li>
		<li>Using scissors, cut remaining tracheo-bronchial tissue along its rings to generate small strips of tissue, and transfer the strips to a fresh Petri dish. Mince the tissue strips using a single sided razor blade to make smaller pieces. 
		NOTE: Since the proximal airways are cartilaginous, they cannot be minced as finely as the distal lung tissue.</li>
		<li>Transfer the minced tissue into the C tubes, add 2 mL of Liberase to the tube ensuring that the tissue is submerged. Load the C tube onto the automated dissociator and run Human Lung Protocol-2 to mechanically dissociate tissue further. 
		NOTE: The dissociator used in this protocol offers an optimized program called human lung protocol-2 for this specific application (see Table of Materials).</li>
	</ol>
	</li>
	<li>Enzyme digestion and single cell isolation
	<ol>
		<li>Transfer approximately 2 g of minced proximal tissue from the C tube into each 50 mL conical centrifuge tube and add 50 &#181;g/mL Liberase and 25 &#181;g/mL DNase solution to each tube. 
		NOTE: To ensure efficient dissociation, tubes should not be filled beyond the 30 mL mark.</li>
		<li>Incubate the minced tissue for 45 min at 37 &#176;C with continuous shaking using a mixer set at 900 rpm.</li>
		<li>Pass the dissociated tissue suspension through a series of cell strainers (500 &#956;m, 300 &#956;m, 100 &#956;m, 70 &#956;m, 40 &#956;m) under vacuum pressure as mentioned above and collect the flow through. Wash the strainer with 20 mL of HBSS+ buffer to collect remaining cells. 
		NOTE: Since proximal tissue is cartilaginous and bulky as compared to the distal tissue, there is a higher possibility of clogging of the filters. Using a funnel can help prevent overflowing of the liquid while passing through the strainers.</li>
		<li>Add an equal volume of HBSS+ buffer to the filtrate to inhibit Liberase activity and prevent over-digestion. Add the isolated luminal proximal airway cells from 3.1.5 to the cell suspension at this step.</li>
		<li>Centrifuge the combined cell suspension at 500 x g for 10 min. Remove the supernatant and repeat the cell wash in HBSS+ buffer. Perform depletion of CD45+ immune cells and CD31+ endothelial cells as mentioned above in 2.4 (optional step).</li>
		<li>Methods for staining is similar to distal lung tissue, follow the steps in&#160; 2.5. Enrich viable epithelial cells based upon their CD45-negative, CD31-negative, CD236-positive cell surface phenotype and negative staining for DAPI.</li>
		<li>Further subset the epithelial cell fraction based upon staining for cell type-specific surface markers, such as NGFR, allowing enrichment of basal (NGFR positive) and non-basal (NGFR negative; secretory, ciliated, neuroendocrine) cell types (Figure 3).</li>
	</ol>
	</li>
</ol>
4. Organoid culture
<ol>
	<li>Add 5,000 (this number can be adjusted to yield the desired density of epithelial organoids) sorted proximal or distal epithelial cells to sterile 1.5 mL tube along with 7.5 x 104 MRC-5 cells (human lung fibroblast cell line). Epithelial-mesenchymal interactions are critical for the expansion of progenitor cells.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C. 
	NOTE: It is important to manually confirm the cell count obtained from the sorter in order to ensure accuracy organoid colony forming efficiency.</li>
	<li>Carefully remove and discard the supernatant and resuspend the cell pellet in 50 &#956;L of ice-cold media supplemented with antibiotics. Keep the cell suspension on ice.</li>
	<li>Add 50 &#956;L of ice cold 1x growth factor depleted basement membrane matrix medium to the vial and gently pipette the suspension on ice to mix. 
	NOTE: It is important to use ice cold media and maintain cells on ice to avoid premature polymerization of the basement membrane matrix medium.</li>
	<li>Transfer the cell suspension onto 0.4 &#181;m&#160;pore-size cell culture insert in a 24 well plate (1.4 x 104 cells/cm2), taking care to avoid introduction of air bubbles.</li>
	<li>Incubate at 37 &#176;C for 30-45 min to allow the matrix to solidify.</li>
	<li>Add 600 &#956;L of pre-warmed growth medium to the well. 
	NOTE: Media was supplemented with antimycotic agents (0.4%) and Pen strep (1%) for the first 24 h after seeding and 10 &#956;M Rho kinase inhibitor for the first 72 h.</li>
	<li>Culture at 37 &#176;C in a 5% CO2 incubator for 30 days, during which time the media should be changed every 48 h. 
	NOTE: The culture duration can be altered based on the purpose of the experiment. Longer endpoints are used to study differentiation whereas shorter endpoints of 7 days, 14 days etc., can be used if the purpose of the experiment is not to achieve complete differentiation.</li>
	<li>Add 10 &#956;M TGF&#946; inhibitor to the culture media for 15 days to maintain the cells in the proliferative phase and suppress overgrowth of fibroblasts. 
	NOTE: Results differ according to the culture medium used for the assay. For e.g., results shown herein were generated using Pneumacult-ALI Medium, which results in the generation of large organoids from distal lung, well differentiated and larger organoids from proximal lung.</li>
</ol>
5. Organoid staining
<ol>
	<li>Fixing and embedding of organoids
	<ol>
		<li>Aspirate media from both the upper and lower transmembrane insert chambers and rinse once with warm PBS.</li>
		<li>Fix the cultures by placing 300 &#181;L of PFA (2% w/v) in the insert and 500 &#181;L in the well for 1hr at 37&#176;C. Remove fixative and rinse with warm PBS taking care not to dislodge the basememt membrane matrix plug. 
		NOTE: Fixed organoids can be stored submerged in PBS at 4 &#176;C for one to two weeks before initiating further steps.</li>
		<li>Aspirate PBS, invert the insert and carefully cut the insert membrane around its periphery. Using forceps, remove transwell membrane, taking care not to disturb the matrix plug.</li>
		<li>In a Petri dish, tap the insert to recover the matrix plug.</li>
		<li>Add a drop of Specimen processing gel such as Histogel (maintained at 37 &#176;C) to the matrix plug and maintain at 4 &#176;C until the gel solidifies.</li>
		<li>Transfer the plug to an embedding cassette, dehydrate through increasing concentrations of ethanol (70, 90 and 100%), clear in xylene and embed in paraffin wax.&#160;</li>
		<li>Cut 7 &#956;m sections on a microtome and collect on positively charged slides.&#160;</li>
	</ol>
	</li>
	<li>Immunofluorescence staining of organoids
	<ol>
		<li>Place slides at 65 &#176;C for 30 min to dewax.&#160;</li>
		<li>Deparaffinize the sections by immersion in xylene and rehydrate through decreasing concentrations of ethanol.</li>
		<li>Perform high temperature antigen retrieval in antigen unmasking solution, citric acid base using a commercially available retriever by dipping slides in the solution for 15 min (Table of Materials).</li>
		<li>Surround the tissue with a hydrophobic barrier using a pap pen.</li>
		<li>Block non-specific staining between the primary antibodies and the tissue, by incubating in Blocking buffer.</li>
		<li>Incubate sections in the appropriate concentration of primary antibodies diluted in incubation solution overnight at 4 &#176;C in a humidified chamber.&#160;</li>
		<li>Rinse sections 3x at room temperature with a washing buffer.</li>
		<li>Incubate in the appropriate concentration of fluorochrome conjugated secondary antibody for 1 h at room temperature.</li>
		<li>Rinse sections 3x at room temperature with 0.1% Tween 20-TBS. Incubate sections for 5 min in DAPI (1 &#181;g/mL). Rinse sections once in TBS (Tris-buffered saline) with 0.1% Tween 20, dry and mount in a mounting solution (Figure 4 and Figure 5). 
		NOTE: Source and optimal working dilution of primary and secondary antibodies used for immunofluorescence staining are included in the Table of Materials.</li>
	</ol>
	</li>
</ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+airway-on-a-chip+enables+analysis+of+human+lung+inflammation+and+drug+responses+in+vitro.">Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro.</a> Nature Methods. 13 (2), 151-157 (2016).</li><li>Huh, 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=Reconstituting+organ-level+lung+functions+on+a+chip.">Reconstituting organ-level lung functions on a chip.</a> Science. 328 (5986), 1662-1668 (2010).</li><li>Jain, 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=Primary+Human+Lung+Alveolus-on-a-chip+Model+of+Intravascular+Thrombosis+for+Assessment+of+Therapeutics.">Primary Human Lung Alveolus-on-a-chip Model of Intravascular Thrombosis for Assessment of Therapeutics.</a> Clinical Pharmacology &amp; Therapeutics. 103 (2), 332-340 (2018).</li><li>Mulay, 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=SARS-CoV-2+infection+of+primary+human+lung+epithelium+for+COVID-19+modeling+and+drug+discovery.">SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery.</a> bioRxiv. , (2020).</li></ol>

Authors have nothing to disclose.

We describe a reliable method for the isolation of defined subpopulations of lung cells from human lung tissue for either molecular or functional analysis and disease modeling. Critical elements of methods include the ability to achieve tissue dissociation with preservation of surface epitopes, which allow antibody-mediated enrichment of freshly isolated cells, and the optimization of culture methods for the efficient generation of region-specific epithelial organoids. We focus on the recovery and enrichment of epithelial progenitor cells capable of forming organoids when recombined with stromal support cells in three-dimensional culture. Even though we did not define the clonality of organoids in these cultures, similar studies performed using isolated mouse lung epithelial progenitor cells were shown to be clonally based upon use of mixed cultures of cells harboring distinct fluorescent reporters22,23.
Methods described herein include adaptations intended to improve cell recovery from digested lung tissue. Digested samples are passed through a 16-gauge needle to further disrupt any remaining undigested clumps and to achieve a homogenous cell suspension. Cell aggregation caused by extruded genomic DNA was mitigated by adding DNase I, which produced a homogeneous cell preparation that provides an uninterrupted fluidics stream during FACS isolation. Together these simple modifications enhance recovery of the target cell populations and avoid delays due to clumping during FACS enrichment.
Previous protocols call for tissue digestion with Elastase, dispase and tripsin/2 mM EDTA to yield a single cell suspension prior to cell isolation4,5. However, this combination of proteases leads to loss of surface proteins and requires that cells are cultured overnight on purcol coated culture dishes for re-expression of surface proteins prior to antibody staining and FACS. By contrast, the combination of Liberase and mechanical agitation to gently disrupt lung tissue provides a more efficient, yet milder, dissociation protocol that can be performed more rapidly while preserving surface epitopes for antibody staining and FACS enrichment. Thus total tissue processing time is condensed and FACS isolation can be performed immediately following tissue dissociation.
These methods allow for the isolation and in vitro culture of epithelial progenitor cells that yield specialized progeny representative of their region of origin. However, these methods can be similarly applied to the identification and enrichment of other cell populations such as immune, vascular and stromal cell types. This could be particularly applicable to the development of regional lung epithelium-on-chip systems which allow for modeling of vascular and epithelial compartments and introduction of other cell types such as immune cells24,25,26.&#160;In a recent study, we used 3D organoid cultures of alveolar epithelium generated using this methodology were used as a tool to study COVID 19 modeling of the lung and&#160; to screen therapeutic targets against SARS-CoV- 2 infection. 27

Airspaces of the human respiratory system can be broadly divided into conducting and respiratory zones that mediate transport of gasses and their subsequent exchange across the epithelial-microvascular barrier, respectively. The conducting airways include trachea, bronchi, bronchioles and terminal bronchioles, whereas respiratory air spaces include respiratory bronchioles, alveolar ducts and alveoli. The epithelial lining of these airspaces changes in composition along the proximo-distal axis to accommodate the unique requirements of each functionally distinct zone. The pseudostratified epithelium of tracheo-bronchial airways is composed of three major cell types, basal, secretory and ciliated, in addition to the less abundant cell types including brush, neuroendocrine and ionocyte1,2,3. Bronchiolar airways harbor morphologically similar epithelial cell types, although there are distinctions in their abundance and functional properties. For example, basal cells are less abundant within bronchiolar airways, and secretory cells include a greater proportion of club cells versus serous and goblet cells that predominate in tracheo-bronchial airways.&#160; Epithelial cells of the respiratory zone include a poorly defined cuboidal cell type in respiratory bronchioles, in addition to alveolar type I (ATI) and type II (ATII) cells of alveolar ducts and alveoli1,4.&#160;
The identity of epithelial stem and progenitor cell types that contribute to the maintenance and renewal of epithelia in each zone are incompletely described and largely inferred from studies in animal models5,6,7,8. Studies in mice have shown that either basal cells &#160;of pseudostratified airways, or club cells of bronchiolar airways or ATII cells of the alveolar epithelium, serve as epithelial stem cells based upon capacity for unlimited self-renewal and multipotent differentiation7,9,10,11,12. Despite the inability to perform genetic lineage tracing studies to assess stemness of human lung epithelial cell types, the availability of organoid-based culture models to assess the functional potential of epithelial stem and progenitor cells provide&#160;a tool for comparative studies between mouse and human13,14,15,16,17.
We describe methods for the isolation of epithelial cell types from different regions of the human lung and their culture using a 3D organoid system to recapitulate the regional cell types.&#160;Similar methods have been developed for the functional analysis and disease modeling of epithelial cells from other organ systems18,19,20,21. These methods provide a platform for the identification of regional epithelial progenitor cells, to perform mechanistic studies investigating their regulation and microenvironment, and to enable disease modeling and drug discovery. Even though studies of lung epithelial progenitor cells performed in animal models can benefit from the analysis, either in vivo or in vitro, insights into the identity of human lung epithelial progenitor cells have been largely dependent upon extrapolation from model organisms. As such, these methods provide a bridge to relate the identity and behavior of human lung epithelial cell types with their studies investigating regulation of stem/progenitor cells.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:694px;" width="694">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Cell Isolation</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">10 mL Sterile syringes, Luer-Lok Tip</td>
			<td style="width:152px;">Fisher scientific</td>
			<td style="width:161px;">BD 309646</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">30 mL Sterile syringes, Luer-Lok Tip</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">BD302832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Biohazard bags</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">89495-440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Biohazard bags</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">89495-440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">connecting ring</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">41-50000-03</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Deoxyribonuclease (lot#SLBF7798V)</td>
			<td style="width:152px;">sigma Aldrich</td>
			<td style="width:161px;">DN25-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Disposable Petri dishes</td>
			<td style="width:152px;">Corning/Falcon</td>
			<td style="width:161px;">25373-187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Funnel</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">42-50000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">HBSS</td>
			<td style="width:152px;">Corning</td>
			<td style="width:161px;">21-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Liberase TM Research Grade</td>
			<td style="width:152px;">sigma Aldrich</td>
			<td style="width:161px;">5401127001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">needle 16G</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">305198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">needle 18G</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">305199</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">PluriStrainer 100 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50100-51</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">PluriStrainer 300 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50300-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">PluriStrainer 40 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50040-51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">PluriStrainer 500 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50500-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">PluriStrainer 70 &#181;m (Cell Strainer)</td>
			<td style="width:152px;">Pluriselect</td>
			<td style="width:161px;">43-50070-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Razor blades</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Red Blood Cell lysis buffer</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:161px;">00-4333-57</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Equipment&#8217;s</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">GentleMACS C Tubes</td>
			<td style="width:152px;">MACS Miltenyi Biotec</td>
			<td style="width:161px;">130-096-334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">GentleMACS Octo Dissociator</td>
			<td style="width:152px;">MACS Miltenyi Biotec</td>
			<td style="width:161px;">130-095-937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Leica ASP 300s Tissue processor</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">LS Columns</td>
			<td style="width:152px;">MACS Miltenyi Biotec</td>
			<td style="width:161px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">MACS MultiStand**</td>
			<td style="width:152px;">Miltenyi Biotech</td>
			<td style="width:161px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Thermomixer</td>
			<td style="width:152px;">Eppendorf</td>
			<td style="width:161px;">05-412-503</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:214px;">Thermomixer</td>
			<td style="width:152px;">Eppendorf</td>
			<td style="width:161px;">05-412-503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS+ Buffer</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:214px;">Amphotericin B</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15290018</td>
			<td style="width:167px;">2ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EDTA (0.5 M), pH 8.0, RNase-free</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">AM9260G</td>
			<td style="width:167px;">500&#181;l</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Fetal Bovine Serum</td>
			<td style="width:152px;">Gemini Bio-Products</td>
			<td style="width:161px;">100-106</td>
			<td style="width:167px;">10ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">HBSS Hank&#39;s Balanced Salt Solution 1X 500 ml</td>
			<td style="width:152px;">VWR</td>
			<td style="width:161px;">45000-456</td>
			<td>500ml bottle</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES (1 M)</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15630080</td>
			<td style="width:167px;">5ml</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:214px;">Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15640055</td>
			<td style="width:167px;">5ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">List of antibodies for FACS</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:214px;">Alexa Fluor 647 anti-human CD326 (EpCAM) Antibody</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">369820</td>
			<td style="width:167px;">1:50</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">BD CompBead Anti-Mouse Ig, K/ Negative control particles set</td>
			<td style="width:152px;">Fisher Scientific</td>
			<td style="width:161px;">BDB552843</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:214px;">CD31 MicroBead Kit, human</td>
			<td style="width:152px;">Miltenyi Biotec</td>
			<td style="width:161px;">130-091-935</td>
			<td style="width:167px;">20&#181;l/ 107 total cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:214px;">CD45 MicroBeads, human</td>
			<td style="width:152px;">Miltenyi Biotec</td>
			<td style="width:161px;">130-045-801</td>
			<td style="width:167px;">20&#181;l/ 107 total cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">DAPI</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:161px;">D9542-10MG</td>
			<td style="width:167px;">1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-human CD235a</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">349104</td>
			<td style="width:167px;">1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-human CD31</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">303104</td>
			<td style="width:167px;">1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-human CD45</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">304054</td>
			<td style="width:167px;">1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-mouse IgM Antibody</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">406506</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Mouse IgM anti human HT2-280</td>
			<td style="width:152px;">Terrace Biotech</td>
			<td style="width:161px;">TB-27AHT2-280</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">PE anti-human CD271(NGFR)</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">345106</td>
			<td style="width:167px;">1:50</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:367px;">Composition of Organoid Culture mediums</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">MRC-5</td>
			<td style="width:152px;">ATCC</td>
			<td style="width:161px;">CCL-171</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">PneumaCult -ALI Medium</td>
			<td style="width:152px;">Stemcell Technologies</td>
			<td style="width:161px;">5001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Small Airway Epithelial Cell Growth Medium</td>
			<td style="width:152px;">PromoCell</td>
			<td style="width:161px;">C-21170</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">ThinCert Tissue Culture Inserts, Sterile</td>
			<td style="width:152px;">Greiner Bio-One</td>
			<td style="width:161px;">662641</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Y-27632 (ROCK inhibitor) 100mM stock (1000x)</td>
			<td style="width:152px;">Stemcell Technologies</td>
			<td style="width:161px;">72302</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Mouse Basal medium:</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Amphotericin B</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15290018</td>
			<td style="width:167px;">50 &#181;l</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">DMEM/F-12, HEPES</td>
			<td style="width:152px;">ThermoFisher scientific</td>
			<td style="width:161px;">11330032</td>
			<td style="width:167px;">50 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Fetal Bovine Serum</td>
			<td style="width:152px;">Gemini Bio-Products</td>
			<td style="width:161px;">100-106</td>
			<td style="width:167px;">5 ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Insulin-Transferrin-Selenium (ITS -G) (100X)</td>
			<td style="width:152px;">ThermoFisher scientific</td>
			<td style="width:161px;">41400045</td>
			<td style="width:167px;">500 &#181;l</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture</td>
			<td style="width:152px;">Thermo fisher scientific</td>
			<td style="width:161px;">15640055</td>
			<td style="width:167px;">500 &#181;l</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">SB431542 TGF-&#946; pathway inhibitor (stock 100 mM)</td>
			<td style="width:152px;">Stem cell</td>
			<td style="width:161px;">72234</td>
			<td style="width:167px;">5 &#181;l</td>
		</tr>
		<tr height="42">
			<td colspan="2" height="42" style="height:42px;width:367px;">List of antibodies for Immunohistochemistry</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Antigen unmasking solution, citric acid based</td>
			<td style="width:152px;">Vector</td>
			<td style="width:161px;">H-3300</td>
			<td style="width:167px;">937 &#181;l in 100ml water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Histogel</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td style="width:161px;">HG-4000-012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Primary Antibodies</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Anti HT2-280</td>
			<td style="width:152px;">Terracebiotech</td>
			<td style="width:161px;">TB-27AHT2-280</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">FOXJ1 Monoclonal Antibody (2A5)</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">14-9965-82</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Human Uteroglobin/SCGB1A1 Antibody</td>
			<td style="width:152px;">R and D systems</td>
			<td style="width:161px;">MAB4218</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Keratin 5 Polyclonal Chicken Antibody, Purified [Poly9059]</td>
			<td style="width:152px;">Biolegend</td>
			<td style="width:161px;">905901</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">MUC5AC Monoclonal Antibody (45M1)</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">MA5-12178</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">PDPN / Podoplanin Antibody (clone 8.1.1)</td>
			<td style="width:152px;">LifeSpan Biosciences</td>
			<td style="width:161px;">LS-C143022-100</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Purified Mouse Anti-E-Cadherin</td>
			<td style="width:152px;">BD biosciences</td>
			<td style="width:161px;">610182</td>
			<td style="width:167px;">1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Sox-2 Antibody</td>
			<td style="width:152px;">Santa Cruz biotechnologies</td>
			<td style="width:161px;">sc-365964</td>
			<td style="width:167px;">1:300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Secondary Antibodies</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Donkey anti-rabbit lgG, 488</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21206</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">FITC anti-mouse IgM Antibody</td>
			<td style="width:152px;">BioLegend</td>
			<td style="width:161px;">406506</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Goat anti-Hamster IgG (H+L), Alexa Fluor 594</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21113</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21121</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21131</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21134</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td style="width:152px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">A-21144</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:214px;">Buffers</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:214px;">Immunohistochemistry Blocking Solution</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;">3% BSA, o.4% Triton-x100 in TBS (Tris based saline)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Immunohistochemistry Incubation Solution</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;">3% BSA, ).1% Triton-X100 in TBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:214px;">Immunohistochemistry Washing Solution</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;">TBS with 0.1% Tween 20</td>
		</tr>
	</tbody>
</table>

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.

Source lung tissue 
The trachea and extrapulmonary bronchus (Figure 1A) were used as the source tissue for isolation of proximal airway epithelial cells and subsequent generation of proximal organoids. Distal lung tissue that includes both parenchyma and small airways of less than 2 mm in diameter (Figure 1A) were used for the isolation of small airway and alveolar epithelial cells (distal lung epithelium) and generation of either small airway or alveolar organoids. Proximal airways lined by a pseudostratified epithelium include abundant basal progenitor cells that are immunoreactive for the membrane protein NGFR (Figure 1B,C). In contrast, epithelial cells lining alveoli included a subset showing apical membrane immunoreactivity with the HTII-280 monoclonal antibody, suggestive of their alveolar type 2 cell (AT2) identity (Figure 1B,D). These surface markers were used to subset single cell suspensions of epithelial cells isolated from either proximal or distal regions.
Tissue dissociation and cell fractionation 
Single cell suspensions of total cells were isolated from either proximal or distal regions of human lung tissue and fractionated using both magnetic bead and FACS to yield enriched epithelial cell populations (Figure 2 and Figure 3). Abundant contaminating cell types including red blood cells, immune cells and endothelial cells were stained using antibodies to CD235a, CD45 and CD31, respectively, followed by magnetic-associated cell sorting&#160; for depletion of these cell types from the total pool of lung cells. The resulting &#8220;depleted&#8221; cell suspensions were significantly enriched for epithelial cell populations in both distal (Figure 2E) and proximal (Figure 3E) tissue samples, with corresponding increase in FACS efficiency. &#160;After depletion of CD235a/CD45/CD31&#160;positive cells using CD45 &#38; CD31 microbeads the percentage of CD31-/CD45-/CD235a- increased from 14% (Figure 2A,B) to 51.7% (Figure 2E,F) in distal population. Further FACS depletion of cells staining positively for either CD235a, CD45 or CD31, elimination of cells with positive staining for DAPI and positive selection for the epithelial cell surface marker CD326, led to a highly enriched distal cell population that accounted for 33.5% (Figure 2E,G) compared to 7% (Figure 2A,C) before depletion of negative population. Further subsetting of distal epithelial cell populations was achieved by fractionation based upon surface staining with the HTII-280 monoclonal antibody (Figure 2D,H), respectively. Accordingly, distal lung epithelial cells included 4.3% HTII-280+ and 2.6% HTII-280- subsets (Figure 2D without depletion&#160;of CD31/CD45/CD235a) and 30% HTII-280+ and 3.6% HTII-280- subsets (Figure 2H after depletion&#160;of CD31/CD45/CD235a).
Total cells isolated from the proximal region were depleted for CD235a/CD45/CD31 positive cells using CD45 &#38; CD31 microbeads and the percentage of CD31-/CD45-/CD235a- increased from 17% (Figure 3A,B) to 56.6% (Figure 3E,F). Positive selection for the epithelial cell surface marker CD326 in cells isolated from the proximal region, led to a highly enriched proximal cell population that accounted for 38% (Figure 3E,G) of total lung cell fractions compared to 9.3% (Figure 3A,C) without depletion of negative population, respectively. Further subsetting of proximal epithelial cell populations was achieved by fractionation based upon surface staining with antibodies to NGFR (Figure 3D,H), respectively. Accordingly, proximal lung epithelial cells included 2.7% NGFR+ and 6.5% NGFR- subsets (Figure 3D without depletion&#160;of CD31/CD45/CD235a) and 13% were NGFR+ and 25% NGFR- (Figure 3H after depletion&#160;of CD31/CD45/CD235a).
Lung organoid cultures 
Distal lung epithelial organoids were cultured within growth-factor depleted basement membrane matrix in media that were empirically tested to optimize for organoid growth and differentiation. Three different media were evaluated including PneumaCult-ALI medium, small airway epithelial cell growth medium (SAECG medium) and mouse Basal medium. Optimal organoid growth was obtained using PneumaCult-ALI medium, which was selected for further studies. Cultures of HTII-280+ distal lung epithelial cells yielded rapidly expanding organoids with an average colony-forming efficiency of 10% (Figure 4A,B). Immunofluorescence staining of day 30 cultures using the HTII-280 and SPC monoclonal antibody revealed lumen-containing organoids composed predominantly of HTII-280+ &#160;and SPC+ distal lung epithelial cells (Figure 4C,C&#8217; and Figure 4D,D&#8217;). Cultures of distal lung epithelial HTII-280- cells yielded organoids that were composed of a pseudostratified epithelium resembling that of small airways (not shown).
Proximal lung epithelial organoids were cultured from NGFR+ cells seeded into Matrigel and cultured for 30 days in PneumaCult-ALI medium. Large lumen-containing organoids were observed (Figure 5D,E,F) with an average colony-forming efficiency of 7.8% (Figure 5A,B,C). Organoids were composed of a pseudostratified epithelium composed of self-renewing Krt5+ and&#160; NGFR+ basal cells (Figure 5D, 5E) and differentiated luminal cell types including FoxJ1+ ciliated cells and MUC5AC+ secretory cells (Figure 5D,E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61541/61541fig1.jpg" /> 
Figure 1: Sampling of human lung tissue. (A) Schematic representation of the human lung showing strategy for sampling proximal and distal regions for cell isolation. (B) H&#38;E staining of the proximal and distal regions of the lung. (C,D) Immunofluorescent staining of corresponding regions showing NGFR+ basal progenitor cells (red) at the basement membrane of bronchial airways and HTII-280+ alveolar type II progenitors (green) in the alveoli. scale bar = 50 &#956;m. <a href="https://www.jove.com/files/ftp_upload/61541/61541fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61541/61541fig2.jpg" /> 
Figure 2: Representative sorting strategy for distal lung cells. (A,E) Percentage of various cell populations before and after depletion of CD45+&#160;and CD31+ population using CD31 and CD45 magnetic beads in distal regions of the lung from one biological sample. (B,F) Representative image of FACS plot showing gating strategy of distal CD31-/CD45-/CD235a- population before and after depletion of CD31/CD45/CD235a positive population (C,G) Epcam+ population before and after depletion of CD31/CD45/CD235a positive population. (D,H) HTII-280+/- population before and after depletion of CD31/CD45/CD235a positive cells. Panels A-D are from the same biological sample and panels E-H are from the same biological sample. <a href="https://www.jove.com/files/ftp_upload/61541/61541fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61541/61541fig3.jpg" /> 
Figure 3: Representative sorting strategy for proximal lung cells. (A,E) Percentage of various cell populations before and after depletion of CD45+&#160;and CD31+ population using CD31 and CD45 magnetic beads in proximal regions of the lung. (B,F) Representative image of FACS plot showing gating strategy of proximal CD31-/CD45-/CD235a- population before and after depletion of CD31/CD45/CD235a positive population (C,G) Epcam+ population before and after depletion of CD31/CD45/CD235a positive population. (D,H) NGFR+/- population before and after depletion of CD31/CD45/CD235a positive cells. Panels A-D and E-H were prepared from two different biological samples. <a href="https://www.jove.com/files/ftp_upload/61541/61541fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61541/61541fig4.jpg" /> 
Figure 4: Characterization of distal lung organoids. (A) Representative image of the human distal organoids cultured in PneumaCult-ALI medium (2x magnification). (B) The Colony forming efficiency (%CFE) was calculated on triplicate wells of organoids derived from two different biological samples. (C, C&#8217;) Immunofluorescent staining of corresponding distal organoids cultured in ALI medium showing HTII-280+ AT2 cells (green). (D, D&#8217;) The marker used for isolation of AT2 cells in this study, HTII-280 costains (green) for the another well characterized AT2 cell marker , SPC (red). Scale bar = 50um. <a href="https://www.jove.com/files/ftp_upload/61541/61541fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61541/61541fig5.jpg" /> 
Figure 5: Characterization of proximal organoids from the human proximal lung. (A,B) Representative image of the human proximal organoids cultured in PneumaCult-ALI medium scale bar 50 &#181;m. (C) The percentage colony forming efficiency (%CFE) was calculated on triplicate wells of organoids derived from two different biological samples. Immunofluorescent staining of differentiated proximal organoids at day 30 with (D) Krt5+ basal cells (green), FoxJ1+ ciliated cells (red) (E) Krt5+ basal cells (green) and MUC5AC+ goblet cells (red). (F) The marker used for isolation of basal cells in this study, NGFR (green) co-stains for the well characterised basal cell marker, Krt5 (red). Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61541/61541fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture at JoVE.com

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

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, ...

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7.9K Views.  Cedars-Sinai Medical Center. 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.

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

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

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 ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

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 ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...