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

Summary

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.

Abstract

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.

Introduction

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 ionocyte^1,2,3. 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 alveoli^1,4. 

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 models^5,6,7,8. 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 differentiation^7,9,10,11,12. 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 human^{13,14,15,16,17}.

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 systems^18,19,20,21. 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. 

Protocol

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

1. Prepare and autoclave all dissection instruments, glassware and the appropriate solutions one day prior to cell isolation.
2. Upon receiving lung tissue, identify and separate the proximal and distal regions. The trachea and bronchi are considered "proximal". For the purposes of this protocol, trachea and the first 2-3 generations of bronchi are disected and used for the isolation of “proximal” airway epithelium. Small airways of 2 mm or less in diameter and surrounding parenchymal tissue are, for the purpose of this protocol, considered as "distal" 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.

2. Enrichment and subsetting of small airway and alveolar epithelial progenitor cells from distal lung tissue

1. Distal tissue preparation
   1. 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.
   2. Wash the tissue 3x with chilled HBSS, discarding HBSS wash each time to remove blood and epithelial lining fluid.
   3. 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.
   4. 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.
2. Enzyme digestion
   NOTE: Liberase stock solution is 5 mg/mL (100x) and DNase stock is 2.5 mg/mL (100x) (Table of Materials).
   1. Add 50 µg/mL Liberase and 25 µg/mL DNase into sterile HBSS in a 50 mL conical tube.
   2. 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 °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.
3. Single cell isolation
   1. 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 μm, 300 μm, 100 μm, 70 μm, 40 μm) under vacuum pressure. Wash the strainer with 20 mL of HBSS+ buffer to collect remaining cells. The recipe for HBSS+ buffer can be found in Table of Materials.
   2. Add an equal volume of HBSS+ buffer, after 45 min to the filtrate to inhibit Liberase activity and prevent over-digestion.
   3. Centrifuge filtrates at 500 x g for 5 min at 4 °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.
   4. Add 10-20 mL of HBSS+ buffer to neutralize RBC lysis buffer. Centrifuge filtrates at 500 x g for 5 min at 4 °C.
   5. If lysed red blood cells (ghost cells) form a cloudy layer above the cell pellet, resuspend pellet in 10 mL of HBSS+ buffer and strain the suspension through 70 μm cell strainer to eliminate the ghost cells. Centrifuge the filtrate at 500 x g for 5 min at 4 °C and proceed with further steps.
4. Depletion of immune cells and endothelial cell (optional step)
   1. Deplete CD31⁺ endothelial cells and CD45⁺ immune cells from the pool of total cells using the CD31 & CD45 microbeads conjugated to monoclonal anti-human CD31 and CD45 antibody (isotype mouse IgG1) and LS columns in accordance to the manufacturer’s protocol (Table of Materials).
   2. 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 °C. Perform a cell count to ascertain the total number of cells in the flow through.
5. Cell surface staining for fluorescence associated cell sorting (FACS)
   1. 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 °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.
   2. Wash cells by adding 3 mL of HBSS+ buffer and centrifuge at 500 x g for 5 min at 4 °C.
   3. 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 °C.
   4. 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 μg/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).

3. Enrichment and subsetting of epithelial progenitor cells from tracheo-bronchial airways

1. Tissue preparation
   1. Dissect out proximal airways (trachea/bronchi) from the lungs. Open airways along their length using scissors to expose the lumen and add 50 µg/mL Liberase to fully cover the tissue.
   2. Incubate for 20 min at 37 °C with continuous shaking using a thermo mixer set at 900 rpm.
   3. 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.
   4. 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. 
   5. Centrifuge the suspension at 500 x g for 5 min at 4 °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.
   6. 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.
   7. 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).
2. Enzyme digestion and single cell isolation
   1. Transfer approximately 2 g of minced proximal tissue from the C tube into each 50 mL conical centrifuge tube and add 50 µg/mL Liberase and 25 µg/mL DNase solution to each tube.
      NOTE: To ensure efficient dissociation, tubes should not be filled beyond the 30 mL mark.
   2. Incubate the minced tissue for 45 min at 37 °C with continuous shaking using a mixer set at 900 rpm.
   3. Pass the dissociated tissue suspension through a series of cell strainers (500 μm, 300 μm, 100 μm, 70 μm, 40 μm) 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.
   4. 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.
   5. 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).
   6. Methods for staining is similar to distal lung tissue, follow the steps in  2.5. Enrich viable epithelial cells based upon their CD45-negative, CD31-negative, CD236-positive cell surface phenotype and negative staining for DAPI.
   7. 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).

4. Organoid culture

1. 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.
2. Centrifuge at 500 x g for 5 min at 4 °C.
   NOTE: It is important to manually confirm the cell count obtained from the sorter in order to ensure accuracy organoid colony forming efficiency.
3. Carefully remove and discard the supernatant and resuspend the cell pellet in 50 μL of ice-cold media supplemented with antibiotics. Keep the cell suspension on ice.
4. Add 50 μL 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.
5. Transfer the cell suspension onto 0.4 µm pore-size cell culture insert in a 24 well plate (1.4 x 10⁴ cells/cm²), taking care to avoid introduction of air bubbles.
6. Incubate at 37 °C for 30-45 min to allow the matrix to solidify.
7. Add 600 μL 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 μM Rho kinase inhibitor for the first 72 h.
8. Culture at 37 °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.
9. Add 10 μM TGFβ 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.

5. Organoid staining

1. Fixing and embedding of organoids
   1. Aspirate media from both the upper and lower transmembrane insert chambers and rinse once with warm PBS.
   2. Fix the cultures by placing 300 µL of PFA (2% w/v) in the insert and 500 µL in the well for 1hr at 37°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 °C for one to two weeks before initiating further steps.
   3. 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.
   4. In a Petri dish, tap the insert to recover the matrix plug.
   5. Add a drop of Specimen processing gel such as Histogel (maintained at 37 °C) to the matrix plug and maintain at 4 °C until the gel solidifies.
   6. 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. 
   7. Cut 7 μm sections on a microtome and collect on positively charged slides. 
2. Immunofluorescence staining of organoids
   1. Place slides at 65 °C for 30 min to dewax. 
   2. Deparaffinize the sections by immersion in xylene and rehydrate through decreasing concentrations of ethanol.
   3. 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).
   4. Surround the tissue with a hydrophobic barrier using a pap pen.
   5. Block non-specific staining between the primary antibodies and the tissue, by incubating in Blocking buffer.
   6. Incubate sections in the appropriate concentration of primary antibodies diluted in incubation solution overnight at 4 °C in a humidified chamber. 
   7. Rinse sections 3x at room temperature with a washing buffer.
   8. Incubate in the appropriate concentration of fluorochrome conjugated secondary antibody for 1 h at room temperature.
   9. Rinse sections 3x at room temperature with 0.1% Tween 20-TBS. Incubate sections for 5 min in DAPI (1 µg/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.

Representative Results

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.

Discussion

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 reporters^22,23.

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 isolation^4,5. 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 cells^24,25,26. 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

Materials

Name	Company	Catalog Number	Comments
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