Method Article

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

DOI:

10.3791/61541

āø±

July 21st, 2020

In This Article

Summary

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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

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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 NGFRĀ 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

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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, bas....

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Protocol

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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 consi....

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Results

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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 airwa.......

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Discussion

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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.......

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Disclosures

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Authors have nothing to disclose.

Acknowledgements

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We appreciate support from Mizuno Takako for IFC and H and E staining, Vanessa Garcia for tissue sectioningĀ  andĀ  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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Cell Isolation
10 mL Sterile syringes, Luer-Lok TipFisher scientificBD 309646
30 mL Sterile syringes, Luer-Lok TipVWRBD302832
Biohazard bagsVWR89495-440
Biohazard bagsVWR89495-440
connecting ringPluriselect41-50000-03
Deoxyribonuclease (lot#SLBF7798V)sigma AldrichDN25-1G
Disposable Petri dishesCorning/Falcon25373-187
FunnelPluriselect42-50000
HBSSCorning21-023
Liberase TM Research Gradesigma Aldrich5401127001
needle 16GVWR305198
needle 18GVWR305199
PluriStrainer 100 µm (Cell Strainer)Pluriselect43-50100-51
PluriStrainer 300 µm (Cell Strainer)Pluriselect43-50300-03
PluriStrainer 40 µm (Cell Strainer)Pluriselect43-50040-51
PluriStrainer 500 µm (Cell Strainer)Pluriselect43-50500-03
PluriStrainer 70 µm (Cell Strainer)Pluriselect43-50070-51
Razor bladesVWR55411-050
Red Blood Cell lysis buffereBioscience00-4333-57
Equipment’s
GentleMACS C TubesMACS Miltenyi Biotec130-096-334
GentleMACS Octo DissociatorMACS Miltenyi Biotec130-095-937
Leica ASP 300s Tissue processor
LS ColumnsMACS Miltenyi Biotec130-042-401
MACS MultiStand**Miltenyi Biotech130-042-303
ThermomixerEppendorf05-412-503
ThermomixerEppendorf05-412-503
HBSS+ Buffer
Amphotericin BThermo fisher scientific152900182ml
EDTA (0.5 M), pH 8.0, RNase-freeThermo fisher scientificAM9260G500µl
Fetal Bovine SerumGemini Bio-Products100-10610ml
HBSS Hank's Balanced Salt Solution 1X 500 mlVWR45000-456500ml bottle
HEPES (1 M)Thermo fisher scientific156300805ml
Penicillin-Streptomycin-Neomycin (PSN) Antibiotic MixtureThermo fisher scientific156400555ml
List of antibodies for FACS
Alexa Fluor 647 anti-human CD326 (EpCAM) AntibodyBioLegend3698201:50
BD CompBead Anti-Mouse Ig, K/ Negative control particles setFisher ScientificBDB552843
CD31 MicroBead Kit, humanMiltenyi Biotec130-091-93520µl/ 107 total cells
CD45 MicroBeads, humanMiltenyi Biotec130-045-80120µl/ 107 total cells
DAPISigma AldrichD9542-10MG1:10000
FITC anti-human CD235aBioLegend3491041:100
FITC anti-human CD31BioLegend3031041:100
FITC anti-human CD45BioLegend3040541:100
FITC anti-mouse IgM AntibodyBioLegend4065061:500
Mouse IgM anti human HT2-280Terrace BiotechTB-27AHT2-2801:300
PE anti-human CD271(NGFR)BioLegend3451061:50
Composition of Organoid Culture mediums
MRC-5ATCCCCL-171
PneumaCult -ALI MediumStemcell Technologies5001
Small Airway Epithelial Cell Growth MediumPromoCellC-21170
ThinCert Tissue Culture Inserts, SterileGreiner Bio-One662641
Y-27632 (ROCK inhibitor) 100mM stock (1000x)Stemcell Technologies72302
Mouse Basal medium:
Amphotericin BThermo fisher scientific1529001850 µl
DMEM/F-12, HEPESThermoFisher scientific1133003250 ml
Fetal Bovine SerumGemini Bio-Products100-1065 ml
Insulin-Transferrin-Selenium (ITS -G) (100X)ThermoFisher scientific41400045500 µl
Penicillin-Streptomycin-Neomycin (PSN) Antibiotic MixtureThermo fisher scientific15640055500 µl
SB431542 TGF-β pathway inhibitor (stock 100 mM)Stem cell722345 µl
List of antibodies for Immunohistochemistry
Antigen unmasking solution, citric acid basedVectorH-3300937 µl in 100ml water
HistogelThermo ScientificHG-4000-012
Primary Antibodies
Anti HT2-280TerracebiotechTB-27AHT2-2801:500
FOXJ1 Monoclonal Antibody (2A5)Thermo Fisher Scientific14-9965-821:300
Human Uteroglobin/SCGB1A1 AntibodyR and D systemsMAB42181:300
Keratin 5 Polyclonal Chicken Antibody, Purified [Poly9059]Biolegend9059011:500
MUC5AC Monoclonal Antibody (45M1)Thermo Fisher ScientificMA5-121781:300
PDPN / Podoplanin Antibody (clone 8.1.1)LifeSpan BiosciencesLS-C143022-1001:300
Purified Mouse Anti-E-CadherinBD biosciences6101821:1000
Sox-2 AntibodySanta Cruz biotechnologiessc-3659641:300
Secondary Antibodies
Donkey anti-rabbit lgG, 488Thermo Fisher ScientificA-212061:500
FITC anti-mouse IgM AntibodyBioLegend4065061:500
Goat anti-Hamster IgG (H+L), Alexa Fluor 594Thermo Fisher ScientificA-211131:500
Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 488Thermo Fisher ScientificA-211211:500
Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 488Thermo Fisher ScientificA-211311:500
Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 568Thermo Fisher ScientificA-211341:500
Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 568Thermo Fisher ScientificA-211441:500
Buffers
Immunohistochemistry Blocking Solution3% BSA, o.4% Triton-x100 in TBS (Tris based saline)
Immunohistochemistry Incubation Solution3% BSA, ).1% Triton-X100 in TBS
Immunohistochemistry Washing SolutionTBS with 0.1% Tween 20

References

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  1. Rackley, C. R., Stripp, B. R. Building and maintaining the epithelium of the lung. Journal of Clinical Investigation. 122 (8), 2724-2730 (2012).
  2. Montoro, D. T., et al. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature.

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Tags

Lung Epithelial Progenitor CellsOrganoid CultureFluorescence Activated Cell SortingTissue DissociationCell Surface MarkersNGFR Basal CellsHTII 280 Alveolar CellsMagnetic Bead DepletionColony Forming Efficiency3D Organoid Generation

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