Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics

Natalie Elia1, Jennifer Lippincott‐Schwartz1

1 Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.22
DOI:  10.1002/0471143030.cb0422s43
Online Posting Date:  June, 2009
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Epithelial cells grown in three‐dimensional (3‐D) cultures of extracellular matrix differentiate into a multicellular structure of polarized cells. This process shares many characteristics with the physiological development of an epithelial tissue and the formation of polarity in epithelial cells. Imaging 3‐D cultures of polarized epithelial cells is therefore a powerful tool to study epithelial architecture and morphogenesis under close‐to‐physiological conditions. The new generation of confocal microscopes allows live‐cell imaging of fluorescently tagged molecules in these cultures. This opens up new opportunities for studying how molecules behave and are distinguished asymmetrically within a 3‐D setting. This unit discusses technical aspects for culturing and imaging MDCK 3‐D culture for both fixed 3‐D cultures and live‐cell imaging. Curr. Protoc. Cell Biol. 43:4.22.1‐4.22.18. © 2009 by John Wiley & Sons, Inc.

Keywords: 3‐D cultures; MDCK cells; live‐cell imaging; MDCK cyst; epithelial cyst; 3‐D culture imaging

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growing MDCK Three‐Dimensional Cultures
  • Basic Protocol 2: Indirect Immunofluorescence of MDCK 3‐D Cultures
  • Basic Protocol 3: Imaging Immunostained 3‐D Cultures
  • Alternate Protocol 1: Live‐Cell Imaging of MDCK 3‐D Cultures
  • Basic Protocol 4: FRAP and FLIP in MDCK 3‐D Cultures
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Growing MDCK Three‐Dimensional Cultures

  • MDCK II cells
  • MEM supplemented with 10% FBS, 2 mM glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin
  • 1 mg/ml of G418, optional
  • 0.25% (w/v) trypsin/1 mM EDTA
  • 200 mM GlutaMAX (GIBCO, Invitrogen) stock (or 200 mM glutamine)
  • 280 mM NaHCO 3 in water (store at 4°C)
  • 10× MEM without glutamine (store at 4°C)
  • 1 M HEPES, pH 7.6
  • 3 mg/ml PureCol purified collagen I (INAMED Biomaterials) or any highly purified bovine collagen I (store at 4°C)
  • Sterile tissue culture hood
  • 10‐cm tissue culture dishes
  • 15‐ml conical tube
  • pH indicator strips
  • 4‐well Lab‐Tek chamber slide (Nalge Nunc cat. no. 155383)
  • 37°C oven
  • Nunc Anapore membrane inserts (0.2‐µm pore, 10‐mm; Nalge Nunc cat. no. 13935)
  • Hemacytometer

Basic Protocol 2: Indirect Immunofluorescence of MDCK 3‐D Cultures

  • 3‐D MDCK cultures on membrane inserts (see protocol 1)
  • 1000 U/ml collagenase‐1 (type CVII, Sigma cat. no. C‐2799) in PBS+ stock (store in aliquots at −80°C)
  • PBS+: 1× PBS supplemented with 0.9 mM Ca2+ and 0.5 mM Mg2+ (store at room temperature)
  • 4% (w/v) paraformaldehyde (PFA)
  • Permeabilization solution: 10% FBS, 0.5% Triton X‐100 in PBS (make fresh for every experiment, store at 4°C during the experiment)
  • Primary antibody against protein of interest
  • Secondary antibody tagged with fluorophore (e.g., Alexa and Cy dyes)
  • Hoechst 33258 nuclear dye (Molecular Probes cat. no. H‐3569)
  • Fluorescently tagged phalloidin (Molecular Probes; Invitrogen)
  • Fluoromount‐G mounting solution (SouthernBiotech cat. no. 0100‐01)
  • Clear nail polish
  • 37°C incubator
  • Platform shaker
  • Microscope slides
  • Carbon steel blades
  • Fine forceps
  • Circular coverslips (18 to 25 mm)

Basic Protocol 3: Imaging Immunostained 3‐D Cultures

  • Immunostained 3‐D culture sample
  • High‐resolution confocal microscope (e.g., either the Olympus F1000 or the Zeiss 510)
  • Oil‐immersion objective: 40× NA 1.3 Plan Neofluar or UPlanFI or 60×/63× NA 1.4 Plan Apochromat oil objective
  • Three‐dimensional rendering software for image analysis of 3‐D culture cysts (e.g., Volocity software from Improvision)

Alternate Protocol 1: Live‐Cell Imaging of MDCK 3‐D Cultures

  • Fluorescently labeled 3‐D MDCK cultures (see protocol 1)
  • Collagen‐coated 4‐well chamber slides (Nunc; see protocol 1)
  • FM4‐64 membrane dye (Invitrogen)
  • 37°C incubator
  • 37°C, 5% CO 2 incubator
  • Confocal microscope (for high‐speed acquisition, spinning disc confocal or the Zeiss LSM live duo system)
  • Water objective (40× or 60× NA1.2)
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Literature Cited

   Debnath, J. and Brugge, J.S. 2005. Modeling glandular epithelial cancers in three‐dimensional cultures. Nat. Rev. Cancer. 5:675‐688.
   Kim, M., Datta, A., Brakeman, P., Yu, W., and Mostov, K.E. 2007. Polarity proteins PAR6 and aPKC regulate cell death through GSK‐3beta in 3D epithelial morphogenesis. J. Cell Sci. 120:2309‐2317.
   Lee, G.Y., Kenny, P.A., Lee, E.H., and Bissell, M.J. 2007. Three‐dimensional culture models of normal and malignant breast epithelial cells. Nat. Methods 4:359‐365.
   Martin‐Belmonte, F., Gassama, A., Datta, A., Yu, W., Rescher, U., Gerke, V., and Mostov, K. 2007. PTEN‐mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42. Cell 128:383‐397.
   Martin‐Belmonte, F. and Mostov, K. 2008. Regulation of cell polarity during epithelial morphogenesis. Curr. Opin. Cell Biol. 20:227‐234.
   Martín‐Belmonte, F., Yu, W., Rodríguez‐Fraticelli, A.E., Ewald, A., Werb, Z., Alonso, M.A., and Mostov, K. 2008. Cell‐polarity dynamics control the mechanism of lumen formation in epithelial morphogenesis. Curr. Biol. 18:507‐513.
   McAteer, J.A., Evan, A.P., and Gardner, K.D. 1987. Morphogenetic clonal growth of kidney epithelial cell line MDCK. Anat. Rec. 217:229‐239.
   O'Brien, L.E., Jou, T.S., Pollack, A.L., Zhang, Q., Hansen, S.H., Yurchenco, P., and Mostov, K.E. 2001. Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly. Nat. Cell Biol. 3:831‐838.
   O'Brien, L.E., Zegers, M.M.P., and Mostov, K.E. 2002. Building epithelial architecture: Insights from three‐dimensional culture models. Nat. Rev. Mol. Cell Biol. 3:531‐537.
   O'Brien, L.E., Yu, W., Tang, K., Jou, T.S., Zegers, M.M., and Mostov, K.E. 2006. Morphological and biochemical analysis of Rac1 in three‐dimensional epithelial cell cultures. Methods Enzymol. 406:676‐691.
   Schmeichel, K.L. and Bissell, M.J. 2003. Modeling tissue‐specific signaling and organ function in three dimensions. J. Cell Sci. 116:2377‐2388.
   Simons, K. and Fuller, S.D. 1985. Cell surface polarity in epithelia. Annu. Rev. Cell Biol. 1:243‐288.
Key Reference
   O'Brien et al., 2006. See above.
  This method paper describes in detail a protocol for plating and growing MDCK cells for immunostaining as well as for biochemical analysis.
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