Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF‐HR9 Endodermal Cells

Israel Vlodavsky1

1 Hadassah‐Hebrew University Hospital, Jerusalem
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.4
DOI:  10.1002/0471143030.cb1004s01
Online Posting Date:  May, 2001
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The ECM is an organized complex of collagens, proteoglycans, and glycoproteins, all interacting to produce a highly stable structure upon which cells migrate, proliferate, differentiate, and survive in vivo. Cultured bovine corneal endothelial (BCE) cells and PF‐HR9 endodermal cells produce underlying ECMs that adhere strongly to plastic and closely resemble subendothelial and subepithelial basement membranes in vivo in their morphology, molecular composition, and biological activities. This unit describes the methods for preparation of these ECMs, their properties, and their cellular effects.

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Table of Contents

  • Basic Protocol 1: Preparation of Bovine Corneal Endothelial Cell ECM (BCE‐ECM)
  • Alternate Protocol 1: Preparation of Metabolically Labelled ECM
  • Alternate Protocol 2: Preparation of HR9‐ECM
  • Support Protocol 1: Cell Attachment Assay
  • Support Protocol 2: Cell Proliferation Assay
  • Support Protocol 3: Cell Differentiation Assay
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Preparation of Bovine Corneal Endothelial Cell ECM (BCE‐ECM)

  • 10 to 20 eyes of freshly slaughtered cows
  • 95% (v/v) ethanol
  • PBS ( appendix 2A)
  • Supplemented low‐glucose DMEM‐10 (see recipe)
  • Recombinant human basic fibroblast growth factor (bFGF; Sigma)
  • Trypsin/EDTA solution (see recipe)
  • Dextran T40 (Sigma)
  • Triton/NH 4OH cell lysis solution (see recipe)
  • Gentamicin (Life Technologies)
  • Fungizone (amphotericin B; Life Technologies)
  • 18‐G needles
  • 10‐cm, 6‐cm, and 35‐mm tissue culture dishes
  • Groove director (curette or spoon‐headed spatula), sterile
  • 37°C, 10% (v/v) CO 2 incubator with 100% humidity
  • 10‐cm gelatin‐coated tissue culture dishes (see recipe)

Alternate Protocol 1: Preparation of Metabolically Labelled ECM

  • PF‐HR9 cells (ATCC; Chung et al., ; Kramer and Vogel, )
  • Supplemented high‐glucose DMEM‐10 (see recipe)
  • Ascorbic acid (Sigma)
  • Penicillin
  • Streptomycin
  • Fibronectin‐coated tissue culture dishes (see recipe)

Alternate Protocol 2: Preparation of HR9‐ECM

  • HS703T cells (Vlodavsky et al., ; Vlodavsky and Gospodarowicz, )
  • Supplemented high‐glucose DMEM‐10 (see recipe)
  • Trypsin/EDTA solution (see recipe)
  • 35‐mm ECM‐coated tissue culture dishes (see protocol 1; see protocol 3)
  • PBS ( appendix 2A)
  • 35‐mm tissue culture dishes
  • Coulter counter

Support Protocol 1: Cell Attachment Assay

  • Aortic tissue from freshly sacrificed cows
  • DMEM/BCS: supplemented low‐glucose DMEM‐10 (see recipe) in which FBS and newborn calf serum are replaced with 10% (v/v) bovine calf serum
  • Trypsin/EDTA solution (see recipe)
  • 35‐mm regular and BCE‐ECM‐coated tissue culture dishes (see protocol 1)
  • 3.7% formaldehyde (prepared from commercial 37% formalin)
  • 0.125% (w/v) crystal violet solution (see recipe)

Support Protocol 2: Cell Proliferation Assay

  • PC12 cells (ATCC)
  • Supplemented high‐glucose DMEM‐10 (see recipe)
  • Trypsin/EDTA solution (see recipe)
  • 35‐mm regular and BCE‐ECM‐coated tissue culture dishes (see protocol 1)
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Literature Cited

Literature Cited
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Key References
   Gospodarowicz et al., 1980a. See above.
  See note for Vlodavsky et al. ().
   Gospodarowicz et al., 1984. See above.
  First article describing the effects of HR9‐ECM on cell proliferation and differentiation.
   Vlodavsky et al., 1980. See above.
  This and Gospodarowicz et al. () are the first two articles describing the preparation, composition, and biological effects (induction of cell attachment, flattening, and proliferation) of BCE‐ECM on normal and malignant cells.
   Vlodavsky et al., 1993. See above.
  Review article emphasizing the biological significance of ECM‐resident bFGF and other active molecules.
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