Name: isolation and enrichment of human lung epithelial progenitor cells for organoid culture
Uploaded: 2020-07-21T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture at JoVE.com

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
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	<li>Prepare and autoclave all dissection instruments, glassware and the appropriate solutions one day p...

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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 epithelia...

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;

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			<td height="21" style="height:21px;width:214px;">Cell Isolation</td>
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			<td height="42" style="height:42px;width:214px;">10 mL Sterile syringes, Luer-Lok Tip</td>
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			<td height="42" style="height:42px;width:214px;">30 mL Sterile syringes, Luer-Lok Tip</td>
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			<td style="width:167px;">2ml</td>
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			<td height="40" style="height:40px;">EDTA (0.5 M), pH 8.0, RNase-free</td>
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			<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 air...

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, ...

3D organoid cultures are proximal in distal lung epithelium developed using this protocol. Provide tractable model to study fundamental mechanisms of lung tissue maintenance or disease associated remodeling, and serves an effective platform for drug discovery and validation. This protocol is compatible with other tissue dissociation cellular fractionation approaches.And since epithelial progenitor cells is isolated from different anatomic compartments maintain their positional identity in vitro, it can be used for modeling regional differences in lung responses to either exogenous or endogenous stimuli. This level technique can be used for the isolation of different subpopulation of epithelial cells including region specific progenitor cells that can be cultured to yield specialist differentiated progeny representative of the region of origin. Upon receive of the lung tissue, separate the proximal trachea and bronchi from the distal lung epithelium, which contains small airways of two millimeters in diameter or less, and the surrounding parenchymal tissue.Place the samples in individual sterile 150 by 15 millimeter Petri dishes and dice the distal tissue sample into approximately one centimeter cubed pieces. Place the pieces into a clean 50 millimeter tube and wash the tissues three times with fresh chilled HBSS per wash. After the last wash, transfer the tissues to a new Petri dish and blot the pieces dry with sterile anti lint wipes.Then use forceps and scissors to remove as much visceral pleura as possible before mincing the tissues into approximately two millimeter diameter fragments. For tissue fragment digestion, add 50 micrograms per milliliter of liberase and 25 micrograms per milliliter of DNAs into 25 milliliters of sterile HBSS in a 50 milliliter conical tube. And add approximately two to three grams of the minced tissue to the tube.Then place the tissues at 37 degrees Celsius with continuous shaking at 900 revolutions per minute for 40 to 60 minutes. After 30 minutes, use a 10 milliliter syringe to triturate the digested tissue to prevent clump formation. At the end of the incubation, use a 10 milliliter syringe equipped with a 16 gauge needle to triturate the tissue five times.Then use a wide bore pipette to pass the suspension through a series of cell strainers under vacuum pressure as indicated. After washing the last strainer with 20 milliliters of HBSS plus to collect any remaining cells, centrifuge the filtrates to collect the isolated cells. After discarding the supernatants, add one milliliter of red blood cell lysis buffer to the pellets with gentle pipetting before incubating the cells for one minute done ice.At the end of the incubation, add 10 to 20 milliliters of HBSS plus buffer to neutralize the lysis and collect the remaining cells by centrifugation. Deplete CD31 positive endothelial cells and CD45 positive immune cells from the pool of total cells using the CD31 and CD45 microbeads conjugated to monoclonal anti-human CD31 and CD45 antibody and LS Columns in accordance to the manufacturers'protocol. For cell surface staining for FACS, add the appropriate primary antibodies of interest at the required concentration and incubate the cells for 30 minutes at four degrees Celsius in the dark.At the end of the incubation, wash the cells with three milliliters of HBSS plus and labeled the cells with the appropriate concentration of fluorophore conjugated secondary antibody for a 30-minute incubation on ice as appropriate. At the end of the incubation, wash the cells with three milliliters of HBSS plus and resuspend the cells at a one times 10 to the seventh cells per milliliter of HBSS plus concentration before filtering the cells into five milliliter polystyrene tubes through a strainer cap to ensure the formation of a single cell suspension. Then add one microgram per milliliter of DAPI to stain the permeable cells.For proximal tissue epithelial progenitor cell enrichment, dissect out the proximal airways from the lungs and use scissors to open the airways along their length to expose the lumen. Cover the tissues in 50 micrograms per milliliter of liberase for 20-minute incubation at 37 degrees Celsius with continuous shaking on a mixer. At the end of the incubation, transfer the tissue to a sterile 150 by 15 millimeter dish and use a scalpel to gently scrape the airway surface to completely strip the luminal epithelial cells from the tissue.Next, wash the dish with five milliliters of sterile HBSS plus buffer to collect all of the dislodged luminal epithelial cells and transfer the cells to a 50 milliliter conical centrifuge tube. Successively triturate the suspension five times using a 10 milliliter pipette to obtain a single cell suspension. Then collect the suspension by centrifugation.Then resuspend the pellet in fresh HBSS plus buffer on ice. Use scissors to cut the remaining tracheobronchial tissue along its rings to generate small strips of tissue. In a new dish, use a single sided razor blade to mince the strips into smaller pieces and transfer the tissue pieces into C tubes containing two milliliters of liberase per tube.Then use human lung protocol two on a gentleMACS automated dissociator to mechanically dissociate the tissue further. At the end of the dissociation, transfer approximately two grams of minced proximal tissue to 50 milliliter conical tubes containing 50 micrograms per milliliter of liberase and 25 micrograms per milliliter of DNA solution. At the end of a 45-minute incubation at 37 degrees Celsius with shaking, filter the tissue slurry through a series of strainers as demonstrated for the distal lung tissue and add an equal volume of HBSS plus buffer to the filtrate to stop the digestion.Add the isolated luminal proximal airway cells to the tube and collect the cells by centrifugation. The cells can then be enriched by microbead isolation and stained for cell surface markers of interest as demonstrated for the distal lung tissue cells. Proximal airways lined by a pseudo stratified epithelium include abundant basal progenitor cells that are immuno reactive for the membrane protein NGFR.In contrast, epithelial cells lining the alveoli include a subset that shows apical membrane immunoreactivity with the HTII-280 monoclonal antibody suggestive of their alveolar type II cell identity. Magnetic bead depletion of contaminating red blood, immune, and endothelial cells results in a significant enrichment of epithelial cell populations in both distal and proximal tissue samples with corresponding increases in FACS efficiency. Further FACS depletion leads to highly enriched distal cell populations that can be additionally fractionated based on their NGFR or HTII-280 surface staining.HTII-280 positive distal lung epithelial cell cultures yield rapidly expanding organoids with an average colony forming efficiency of 10%Immunofluorescence staining of day 30 cultures reveals that the lumen containing organoids are composed predominantly of HTII-280 and SPC positive distal lung epithelial cells. Proximal lung epithelial organoids cultured from NGFR positive cells results in the formation of lumen containing organoids at day 30 of culture with an average colony forming efficiency of 7.8%Empirical optimization of enzymatic tissue association is critical for preservation of surface epitopes allowing immunofluorescence staining and enrichment of viable epithelial cell subsets for subsequent generation of region specific organized cultures. This method can be adapted for the identification and enrichment of immune, vascular, stromal, and epithelial cell subsets and development of co-culture models using either 3D organoid chip platforms.We have recently described the use of alveolar organoids generated using this technique as a platform to study SARS-CoV-2 infection of the human lung epithelium and for COVID-19 drug validation.

Research

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Biology

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

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

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Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

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Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

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 ...

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 ...

Chromatin Isolation by RNA Purification (ChIRP)

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 ...

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

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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 ...

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 ...