Preparation of Gelled Substrates

Hynda K. Kleinman1

1 National Institute of Dental Research/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.3
DOI:  10.1002/0471143030.cb1003s00
Online Posting Date:  May, 2001
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Abstract

Gelled substrates can be used for a variety of in vitro and in vivo experiments. A type I collagen gelled substrate will promote cell growth and differentiation. Gelled Matrigel substrate promotes the survival of explanted cells and tissues and the differentiation of a variety of epithelial and endothelial cell types in vitro and to assess angiogenesis and increase tumor growth in vivo. Preparation of the matrices and their use are described in this unit.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Type I Collagen Substrates
  • Basic Protocol 2: Preparation of Gelled Matrigel Substrates
  • Alternate Protocol 1: Growth of Cells Inside Matrigel
  • Alternate Protocol 2: Use of Matrigel In Vivo for Angiogenic Assays and Tumor Growth
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Type I Collagen Substrates

  Materials
  • 3 to 5 mg/ml type I collagen in dilute acid (e.g., Vitrogen 100, Collagen), ice cold
  • 10× phosphate‐buffered saline, pH 7.4 (10× PBS; appendix 2A) or 10× medium salts (e.g., Medium 199, Life Technologies), ice cold
  • 0.1 M NaOH, ice cold
  • 0.1 M HCl, ice cold
  • Dilute acetic acid: 28.5 ml glacial acetic acid/liter
  • Sterile deionized H 2O
  • Cells
  • Tissue culture medium, 37°C
  • Wet ice in a bucket
  • Culture dishes

Basic Protocol 2: Preparation of Gelled Matrigel Substrates

  Materials
  • Matrigel (unit 10.2; Matrigel can also be obtained commercially from Sigma or Becton Dickinson Labware)
  • Medium salts, 4°C (optional, for making weak gels)
  • Cells
  • Tissue culture medium, 37°C
  • Wet ice in a bucket
  • Culture dishes

Alternate Protocol 1: Growth of Cells Inside Matrigel

  Materials
  • Matrigel (unit 10.2; Matrigel can also be obtained commercially from Sigma or Becton Dickinson Labware)
  • Cells
  • Tissue culture medium, 37°C
  • Wet ice in a bucket
  • Culture dishes

Alternate Protocol 2: Use of Matrigel In Vivo for Angiogenic Assays and Tumor Growth

  Materials
  • Matrigel (unit 10.2; Matrigel can also be obtained commercially from Sigma or Becton Dickinson Labware)
  • C57BL6 mice for angiogenesis assay or athymic nude mice for human tumor growth
  • Test compound for angiogenesis assay or tumor sample
  • Proteases (e.g., trypsin or collagenase) for dissociating tumor pieces, if necessary
  • Hemoglobin assay kit (e.g., Drabkin Reagent Kit, Sigma) or additional materials and equipment for histology, including image processor (e.g., NIH Image)
  • Wet ice in a bucket
  • Culture dishes
  • 1‐ to 3‐ml syringes
  • 23‐ or 25‐G needles
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Care and Use Committee (IACUL) and must follow officially approved procedures for the care and use of laboratory animals.
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Figures

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Literature Cited

Literature Cited
   Bendayan, M., Duhr, M.A., and Gingros, D. 1986. Studies on pancreatic acinar cells in tissue cultures: Basal laminin (basement membrane) matrix promotes three‐dimensional reorganization. Eur. J. Cell Biol. 42:60‐67.
   Ben‐Ze'ev, A., Robinson, G.S., Bucher, N.R.L., and Farmer, S.R. 1988. Cell‐cell and cell‐matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 856:2161‐2165.
   Bilozur, M.E. and Hay, E.D. 1988. Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin and collagen. Dev. Biol. 125:19‐33.
   Blau, H., Guzowski, D.E., Siddiqi, Z.A., Scarpelli, E.M., and Bienkowski, R.S. 1988. Fetal type 2 pneumocytes form alveolar‐like structures and maintain long‐term differentiation on extracellular matrix. J. Cell. Physiol. 136:203‐214.
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   Cid, M.C., Grant, D.S., Hoffman, G.S., Auerbach, R., Fauci, A.S., and Kleinman, H.K. 1993. Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis. J. Clin. Invest. 91:977‐985.
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   Fridman, R., Kibbey, M.C., Royce, L.S., Zain, M., Sweeney, T.M., Jicha, D.L., Yannelli, J.R., Martin, G.R., and Kleinman, H.K. 1991. Basement membrane (Matrigel) enhances both the incidence and the growth of subcutaneously injected human and murine cells. J. Natl. Cancer Inst. 83:769‐774.
   Greenberg, G. and Hay, E.D. 1986. Transformation of thyroid epithelium to mesenchyme‐like cells in vitro: Changes in intermediate filament expression and cytodifferentiation. J. Cell Biol. 105:250a.
   Hadley, M.A., Byers, S.W., Suarez‐Quian, C.A., Kleinman, H.K., and Dym, M. 1985. Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation and germ development. J. Cell Biol. 101:1511‐1522.
   Hauschka, S.D. and Konigsberg, I.R. 1966. The influence of collagen on the development of muscle colonies. Proc. Natl. Acad. Sci. U.S.A. 55:119‐126.
   Hoffman, M.P., Kibbey, M.C., Letterio, J.J., and Kleinman, H.K. 1996. Role of laminin‐1 and TGFβ3 in acinar differentiation of a human submandibular salivary gland cell line. J. Cell Sci. 109:2103‐2021.
   Joshi, M.S. 1991. Growth and differentiation of the cultured secretory cells of the cow oviduct on reconstituted basement membrane. J. Exp. Zool. 260:229‐238.
   Kleinman, H.K., Klebe, R.J., and Martin, G.R. 1981. Role of collagenous matrices in the adhesion and growth of cells. J. Cell. Biol. 88:473‐485.
   Kleinman, H.K., McGarvey, M.L., Hassell, J.R., Star, V.L., Cannon, F.B., Laurie, G.W., and Martin, G.R. 1986. Basement membrane complexes with biological activity. Biochemistry 25:312‐318.
   Kleinman, H.K., Graf, J., Iwamoto, Y., Kitten, G.T., Ogle, R.C., Sasaki, M., Yamada, Y., Martin, G.R., and Luckenbill‐Edds, L. 1987. Role of basement membranes in cell differentiation. Ann. N.Y. Acad. Sci. 513:134‐145.
   Kubota, Y., Kleinman, H.K., Martin, G.R., and Lawley, T.J. 1988. Role of laminin and basement membrane in the differentiation of human endothelial cells into capillary‐like structures. J. Cell Biol. 107:1589‐1597.
   Li, L., Aggeler, M.J., Farson, D.A., Hatier, C., Hassell, J.R., and Bissell, M.J. 1986. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 84:136‐140.
   Passaniti, A., Taylor, R.M., Pili, R., Guo, Y., Long, P.V., Haney, J.A., Pauly, R.R., Grant, D.S., and Martin, G.R. 1992. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin and fibroblast growth factor. Lab. Invest. 67:519‐528.
   Taub, M., Wang, Y., Szczesny, T.M., and Kleinman, H.K. 1990. Epidermal growth factor or transforming growth factor alpha is required for kidney tubulogenesis in Matrigel cultures in serum‐free medium. Proc. Natl. Acad. Sci. U.S.A. 87:4002‐4006.
   Vukicevic, S., Luyten, F.P., Kleinman, H.K., and Reddi, A.H. 1990. Differentiation of canaliculi‐like bone network by basement membrane matrix components and defined domains of laminin. Cell 63:437‐445.
   Vukicevic, S., Kleinman, H.K., Luyten, F.P., Roberts, A.B., Roche, N.S., and Reddi, A.H. 1992. Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp. Cell Res. 202:1‐8.
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