Production of 3‐D Airway Organoids From Primary Human Airway Basal Cells and Their Use in High‐Throughput Screening

Marc Hild1, Aron B. Jaffe1

1 Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit IE.9
DOI:  10.1002/cpsc.1
Online Posting Date:  May, 2016
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Abstract

The ability of human airway basal cells to serve as progenitor cells in the conducting airway makes them an attractive target in a number of respiratory diseases associated with epithelial remodeling. This unit describes a protocol for the culture of ‘bronchospheres’, three‐dimensional (3‐D) organoids that are derived from primary human airway basal cells. Mature bronchospheres are composed of functional multi‐ciliated cells, mucin‐producing goblet cells, and airway basal cells. In contrast to existing methods used for the culture of well‐differentiated human airway epithelial cells, bronchospheres do not require growth on a permeable support and can be cultured in 384‐well assay plates. The system provides a mechanism for investigating the regulation of basal cell fate during airway epithelial morphogenesis, as well as a basis for studying the function of the human airway epithelium in high‐throughput assays. © 2016 by John Wiley & Sons, Inc.

Keywords: bronchosphere; ciliated cell; goblet cell; high throughput screening; mucociliary

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Expansion of Human Airway Basal Cells and Bronchosphere Culture
  • Basic Protocol 2: Immunostaining and Imaging of Bronchospheres
  • Basic Protocol 3: Rna Isolation and Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Expansion of Human Airway Basal Cells and Bronchosphere Culture

  Materials
  • Bronchial Epithelial Basal Medium (BEBM; Lonza, cat. no. CC‐3170)
  • BEGM SingleQuot supplements and growth factors kit (Lonza, cat. no. CC‐4175)
  • Human airway basal cells (e.g., Normal Human Bronchial Epithelial Cells; Lonza, cat. no. CC‐2540)
  • 0.05% trypsin‐EDTA (Life Technologies, cat. no. 25300‐054)
  • Trypsin Neutralizing Solution (Lonza, cat. no. CC‐5002)
  • Cell freezing medium (see recipe)
  • Matrigel (Growth Factor Reduced; BD, cat. no. 354230)
  • Differentiation medium (see recipe)
  • Sterile H 2O (cell culture grade)
  • 150‐cm2 cell culture flasks (Corning, cat. no. 430825)
  • Vacuum aspirator
  • 2‐ml disposable aspirating pipets (Falcon cat. no. 357558)
  • Cryovials
  • 384‐well clear‐bottom plastic plates (Greiner, cat. no. 781091)
  • Multichannel pipettor (e.g., Thermo Scientific, cat. no. 2061) and disposable medium reservoirs (VWR, cat. no. 613‐2857)
  • Centrifuge for tubes and assay plates
  • Humidified plate lids (e.g., MicroClime Environmental Lid, Labcyte, cat. no. LL‐0310)

Basic Protocol 2: Immunostaining and Imaging of Bronchospheres

  Materials
  • 20% paraformaldehyde solution (Electron Microscopy Sciences, cat. no. 15713‐S)
  • Live, human airway basal cell‐derived bronchospheres in 384‐well plate ( protocol 1)
  • Immunofluorescence (IF) wash buffer (see recipe)
  • 20% (v/v) goat serum (Sigma, cat. no. G9023‐10 ml) in IF wash buffer
  • Primary and secondary antibodies (Table 1.9.1)
  • Rhodamine‐conjugated phalloidin (Life Technologies, cat. no. R415)
  • Hoechst 33342 (Life Technologies, cat. no. H3570)
  • Phosphate‐buffered saline (PBS; Thermo Fisher Scientific, cat. no. 14190‐144)
  • 25‐ml reagent reservoirs (VistaLab, cat. no. 3054‐1005)
  • BioTek plate washer
  • Vacuum aspirator
  • 2‐ml disposable aspirating pipets (Falcon cat. no. 357558)
  • Seal & Sample aluminum foil lids (Beckman Coulter, cat. no. 538619)

Basic Protocol 3: Rna Isolation and Analysis

  Materials
  • Lysis Mixture (Affymetrix/Panomics, cat. no. 10187–1.7 liters–bulk)
  • Proteinase K (Affymetrix/Panomics, cat. no. 10231–24 ml–bulk)
  • Blocking Reagent (Affymetrix/Panomics, cat. no. 13256–140 ml–bulk)
  • Pooled Probe Sets (Affymetrix/Panomics 8‐plex, HT9000 High Throughput Gene Expression Custom Manufacturing–345 ml)
  • 64 beads—8 × 8–plex format (Affymetrix/Panomics)—HT9000 High Throughput Gene Expression Custom Manufacturing, refer to Table 1.9.2
  • Wash Buffer Component 1 (Affymetrix/Panomics, cat. no. 10205 ‐ 33 ml ‐ bulk)
  • Wash Buffer Component 2 (Affymetrix/Panomics, cat. no. 10193 ‐ 545 ml ‐ bulk)
  • Label Probe Diluent (Affymetrix/Panomics, cat. no. 10676–765 ml–bulk)
  • 2.0 Pre‐Amplifier (Affymetrix/Panomics, cat. no. 15533–4 ml–bulk)
  • 2.0 Amplifier (Affymetrix/Panomics, cat. no. 15532–4 ml–bulk)
  • Label Probe (Affymetrix/Panomics, cat. no. 10940–4 ml–bulk)
  • SAPE (Affymetrix/Panomics, cat. no. 10659–2 ml–bulk)
  • Live, human airway basal cell‐derived bronchospheres ( protocol 1), day 14
  • Sheath fluid (Luminex, cat. no. 40‐50000)
  • Biomek FX with 384‐well head and 384‐well magnet (Beckman Coulter, cat no. A31844, 719715)
  • Multidrop 384 Reagent Dispenser (Thermo Scientific, cat. no. 5840157)
  • 384 well V‐bottom polypropylene plates (Greiner, cat. no. 781280)
  • Bath sonicator
  • PlateLoc Thermal Microplate Sealer (Agilent, cat. no. 01867‐201)
  • Orbital shaking humidified incubator (Liconic, cat. no. 9118 07 00, 9118 12 41, 9121 00 83, 9118 11 02, or 9118 11 16)
  • 384‐well flat clear‐bottom plate (NUNC, cat. no. 242757)
  • ELX406 plate washer with 384‐well magnet (BioTek, cat. no. 406PSUB3‐LB, 7180011, 7102215)
  • Flexmap 3D (Luminex, cat. no. 40‐014)
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Figures

Videos

Literature Cited

Literature Cited
  Araya, J., Cambier, S., Markovics, J.A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W., Jones, K., Broaddus, V.C., Sheppard, D., Barzcak, A., Xiao, Y., Erle, D.J., and Nishimura, S.L. 2007. Squamous metaplasia amplifies pathologic epithelial‐mesenchymal interactions in COPD patients. J. Clin. Invest. 117:3551‐3562. doi: 10.1172/JCI32526.
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  Debnath, J., Muthuswamy, S.K., and Brugge, J.S. 2003. Morphogenesis and oncogenesis of MCF‐10A mammary epithelial acini grown in three‐dimensional basement membrane cultures. Methods 30:256‐268. doi: 10.1016/S1046‐2023(03)00032‐X.
  Fulcher, M.L., Gabriel, S., Burns, K.A., Yankaskas, J.R., and Randell, S.H. 2005. Well‐differentiated human airway epithelial cell cultures. Methods Mol. Med. 107:183‐206. doi: 10.1385/1‐59259‐861‐7:183.
  Gray, T.E., Guzman, K., Davis, C.W., Abdullah, L.H., and Nettesheim, P. 1996. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 14:104‐112. doi: 10.1165/ajrcmb.14.1.8534481.
  Horani, A., Nath, A., Wasserman, M.G., Huang, T., and Brody, S.L. 2013. Rho‐associated protein kinase inhibition enhances airway epithelial basal‐cell proliferation and lentivirus transduction. Am. J. Respir. Cell Mol. Biol. 49:341‐347. doi: 10.1165/rcmb.2013‐0046TE.
  Jaffe, A.B., Kaji, N., Durgan, J., and Hall, A. 2008. Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis. J. Cell Biol. 183:625‐633. doi: 10.1083/jcb.200807121.
  Suprynowicz, F.A., Upadhyay, G., Krawczyk, E., Kramer, S.C., Hebert, J.D., Liu, X., Yuan, H., Cheluvaraju, C., Clapp, P.W., Boucher, R.C., Jr., Kamonjoh, C.M., Randell, S.H., and Schlegel, R. 2012. Conditionally reprogrammed cells represent a stem‐like state of adult epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 109:20035‐20040. doi: 10.1073/pnas.1213241109.
  You, Y. and Brody, S.L. 2013. Culture and differentiation of mouse tracheal epithelial cells. Methods Mol. Biol. 945:123‐143. doi: 10.1007/978‐1‐62703‐125‐7_9.
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