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, 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 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.
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. 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.
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 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 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. 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.
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
2. Enrichment and subsetting of small airway and alveolar epithelial progenitor cells from distal lung tissue
3. Enrichment and subsetting of epithelial progenitor cells from tracheo-bronchial airways
4. Organoid culture
5. Organoid staining
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 for depletion of these cell types from the total pool of lung cells. The resulting “depleted” 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. After depletion of CD235a/CD45/CD31 positive cells using CD45 & 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 of CD31/CD45/CD235a) and 30% HTII-280+ and 3.6% HTII-280– subsets (Figure 2H after depletion of CD31/CD45/CD235a).
Total cells isolated from the proximal region were depleted for CD235a/CD45/CD31 positive cells using CD45 & 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 of CD31/CD45/CD235a) and 13% were NGFR+ and 25% NGFR– (Figure 3H after depletion 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+ and SPC+ distal lung epithelial cells (Figure 4C,C’ and Figure 4D,D’). 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 NGFR+ basal cells (Figure 5D, 5E) and differentiated luminal cell types including FoxJ1+ ciliated cells and MUC5AC+ secretory cells (Figure 5D,E).
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&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 μm. Please click here to view a larger version of this figure.
Figure 2: Representative sorting strategy for distal lung cells. (A,E) Percentage of various cell populations before and after depletion of CD45+ 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. Please click here to view a larger version of this figure.
Figure 3: Representative sorting strategy for proximal lung cells. (A,E) Percentage of various cell populations before and after depletion of CD45+ 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. Please click here to view a larger version of this figure.
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’) Immunofluorescent staining of corresponding distal organoids cultured in ALI medium showing HTII-280+ AT2 cells (green). (D, D’) 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. Please click here to view a larger version of this figure.
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 µm. (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 µm. Please click here to view a larger version of this figure.
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. 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 to screen therapeutic targets against SARS-CoV- 2 infection. 27
The authors have nothing to disclose.
We appreciate support from Mizuno Takako for IFC and H and E staining, Vanessa Garcia for tissue sectioning and 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.
Cell Isolation | |||
10 mL Sterile syringes, Luer-Lok Tip | Fisher scientific | BD 309646 | |
30 mL Sterile syringes, Luer-Lok Tip | VWR | BD302832 | |
Biohazard bags | VWR | 89495-440 | |
Biohazard bags | VWR | 89495-440 | |
connecting ring | Pluriselect | 41-50000-03 | |
Deoxyribonuclease (lot#SLBF7798V) | sigma Aldrich | DN25-1G | |
Disposable Petri dishes | Corning/Falcon | 25373-187 | |
Funnel | Pluriselect | 42-50000 | |
HBSS | Corning | 21-023 | |
Liberase TM Research Grade | sigma Aldrich | 5401127001 | |
needle 16G | VWR | 305198 | |
needle 18G | VWR | 305199 | |
PluriStrainer 100 µm (Cell Strainer) | Pluriselect | 43-50100-51 | |
PluriStrainer 300 µm (Cell Strainer) | Pluriselect | 43-50300-03 | |
PluriStrainer 40 µm (Cell Strainer) | Pluriselect | 43-50040-51 | |
PluriStrainer 500 µm (Cell Strainer) | Pluriselect | 43-50500-03 | |
PluriStrainer 70 µm (Cell Strainer) | Pluriselect | 43-50070-51 | |
Razor blades | VWR | 55411-050 | |
Red Blood Cell lysis buffer | eBioscience | 00-4333-57 | |
Equipment’s | |||
GentleMACS C Tubes | MACS Miltenyi Biotec | 130-096-334 | |
GentleMACS Octo Dissociator | MACS Miltenyi Biotec | 130-095-937 | |
Leica ASP 300s Tissue processor | |||
LS Columns | MACS Miltenyi Biotec | 130-042-401 | |
MACS MultiStand** | Miltenyi Biotech | 130-042-303 | |
Thermomixer | Eppendorf | 05-412-503 | |
Thermomixer | Eppendorf | 05-412-503 | |
HBSS+ Buffer | |||
Amphotericin B | Thermo fisher scientific | 15290018 | 2ml |
EDTA (0.5 M), pH 8.0, RNase-free | Thermo fisher scientific | AM9260G | 500µl |
Fetal Bovine Serum | Gemini Bio-Products | 100-106 | 10ml |
HBSS Hank's Balanced Salt Solution 1X 500 ml | VWR | 45000-456 | 500ml bottle |
HEPES (1 M) | Thermo fisher scientific | 15630080 | 5ml |
Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture | Thermo fisher scientific | 15640055 | 5ml |
List of antibodies for FACS | |||
Alexa Fluor 647 anti-human CD326 (EpCAM) Antibody | BioLegend | 369820 | 1:50 |
BD CompBead Anti-Mouse Ig, K/ Negative control particles set | Fisher Scientific | BDB552843 | |
CD31 MicroBead Kit, human | Miltenyi Biotec | 130-091-935 | 20µl/ 107 total cells |
CD45 MicroBeads, human | Miltenyi Biotec | 130-045-801 | 20µl/ 107 total cells |
DAPI | Sigma Aldrich | D9542-10MG | 1:10000 |
FITC anti-human CD235a | BioLegend | 349104 | 1:100 |
FITC anti-human CD31 | BioLegend | 303104 | 1:100 |
FITC anti-human CD45 | BioLegend | 304054 | 1:100 |
FITC anti-mouse IgM Antibody | BioLegend | 406506 | 1:500 |
Mouse IgM anti human HT2-280 | Terrace Biotech | TB-27AHT2-280 | 1:300 |
PE anti-human CD271(NGFR) | BioLegend | 345106 | 1:50 |
Composition of Organoid Culture mediums | |||
MRC-5 | ATCC | CCL-171 | |
PneumaCult -ALI Medium | Stemcell Technologies | 5001 | |
Small Airway Epithelial Cell Growth Medium | PromoCell | C-21170 | |
ThinCert Tissue Culture Inserts, Sterile | Greiner Bio-One | 662641 | |
Y-27632 (ROCK inhibitor) 100mM stock (1000x) | Stemcell Technologies | 72302 | |
Mouse Basal medium: | |||
Amphotericin B | Thermo fisher scientific | 15290018 | 50 µl |
DMEM/F-12, HEPES | ThermoFisher scientific | 11330032 | 50 ml |
Fetal Bovine Serum | Gemini Bio-Products | 100-106 | 5 ml |
Insulin-Transferrin-Selenium (ITS -G) (100X) | ThermoFisher scientific | 41400045 | 500 µl |
Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture | Thermo fisher scientific | 15640055 | 500 µl |
SB431542 TGF-β pathway inhibitor (stock 100 mM) | Stem cell | 72234 | 5 µl |
List of antibodies for Immunohistochemistry | |||
Antigen unmasking solution, citric acid based | Vector | H-3300 | 937 µl in 100ml water |
Histogel | Thermo Scientific | HG-4000-012 | |
Primary Antibodies | |||
Anti HT2-280 | Terracebiotech | TB-27AHT2-280 | 1:500 |
FOXJ1 Monoclonal Antibody (2A5) | Thermo Fisher Scientific | 14-9965-82 | 1:300 |
Human Uteroglobin/SCGB1A1 Antibody | R and D systems | MAB4218 | 1:300 |
Keratin 5 Polyclonal Chicken Antibody, Purified [Poly9059] | Biolegend | 905901 | 1:500 |
MUC5AC Monoclonal Antibody (45M1) | Thermo Fisher Scientific | MA5-12178 | 1:300 |
PDPN / Podoplanin Antibody (clone 8.1.1) | LifeSpan Biosciences | LS-C143022-100 | 1:300 |
Purified Mouse Anti-E-Cadherin | BD biosciences | 610182 | 1:1000 |
Sox-2 Antibody | Santa Cruz biotechnologies | sc-365964 | 1:300 |
Secondary Antibodies | |||
Donkey anti-rabbit lgG, 488 | Thermo Fisher Scientific | A-21206 | 1:500 |
FITC anti-mouse IgM Antibody | BioLegend | 406506 | 1:500 |
Goat anti-Hamster IgG (H+L), Alexa Fluor 594 | Thermo Fisher Scientific | A-21113 | 1:500 |
Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | A-21121 | 1:500 |
Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | A-21131 | 1:500 |
Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 | Thermo Fisher Scientific | A-21134 | 1:500 |
Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 | Thermo Fisher Scientific | A-21144 | 1:500 |
Buffers | |||
Immunohistochemistry Blocking Solution | 3% BSA, o.4% Triton-x100 in TBS (Tris based saline) | ||
Immunohistochemistry Incubation Solution | 3% BSA, ).1% Triton-X100 in TBS | ||
Immunohistochemistry Washing Solution | TBS with 0.1% Tween 20 |