Use of Hyaluronan‐Derived Hydrogels for Three‐Dimensional Cell Culture and Tumor Xenografts

Monica A. Serban1, Anna Scott2, Glenn D. Prestwich1

1 Department of Medicinal Chemistry and Center for Therapeutic Biomaterials, The University of Utah, Salt Lake City, Utah, 2 Glycosan BioSystems, Salt Lake City, Utah
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.14
DOI:  10.1002/0471143030.cb1014s40
Online Posting Date:  September, 2008
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Abstract

The practice of in vitro three‐dimensional (3‐D) cell culture has lagged behind the realization that classical two‐dimensional (2‐D) culture on plastic surfaces fails to mirror normal cell biology. Biologically, a complex network of proteins and proteoglycans that constitute the extracellular matrix (ECM) surrounds every cell. To recapitulate the normal cellular behavior, scaffolds (ECM analogs) that reconstitute the essential biological cues are required. This unit describes the 3‐D cell culture and tumor engineering applications of Extracel, a novel semisynthetic ECM (sECM), based on cross‐linked derivatives of hyaluronan and gelatin. A simplified cell encapsulation and pseudo‐3‐D culturing (on top of hydrogels) protocol is provided. In addition, the use of this sECM as a vehicle to obtain tumor xenografts with improved take rates and tumor growth is presented. These engineered tumors can be used to evaluate anticancer therapies under physiologically relevant conditions. Curr. Protoc. Cell Biol. 40:10.14.1‐10.14.21. © 2008 by John Wiley & Sons, Inc.

Keywords: hyaluronan; semisynthetic extracellular matrix; hydrogel; biodegradable scaffold

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Standard HA‐Derived Hydrogel Preparation
  • Basic Protocol 2: HA‐Derived Hydrogel Stiffness Variation
  • Basic Protocol 3: ECM Component Incorporation in Hydrogels
  • Basic Protocol 4: Cell Growth on HA‐Derived Hydrogel Surface
  • Basic Protocol 5: Cell Encapsulation in HA‐Derived Hydrogels
  • Basic Protocol 6: HA‐Derived Hydrogels for Tumor Xenografts
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Standard HA‐Derived Hydrogel Preparation

  Materials
  • 7.5‐ml Extracel Hydrogel Kit (Glycosan BioSystems) containing:
    • Glycosil (three 1‐ml vials)
    • Gelin‐S (three 1‐ml vials)
    • Extralink (three 0.5‐ml vials)
    • DG Water (one 10‐ml vial)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Serum‐free cell culture medium
  • 37°C water bath
  • 1‐ml syringes with long‐tip 20‐G × 1½‐in. needles, sterile
  • 37°C shaking or rocking incubator
  • 4‐ml glass vials

Basic Protocol 2: HA‐Derived Hydrogel Stiffness Variation

  Materials
  • 7.5‐ml Extracel Hydrogel Kit (Glycosan BioSystems) containing:
    • Glycosil (three 1‐ml vials)
    • Gelin‐S (three 1‐ml vials)
    • Extralink (three 0.5‐ml vials; purchase additional vials separately, if required)
    • DG Water (one 10‐ml vial)
  • Serum‐free cell culture medium
  • Phosphate‐buffered saline (PBS; appendix 2A), pH ∼7.4 and ∼7.6
  • 37°C shaking or rocking incubator
  • 37°C water bath
  • 1‐ml syringes with long‐tip 20‐G × 1½‐in. needles, sterile

Basic Protocol 3: ECM Component Incorporation in Hydrogels

  Materials
  • 1‐ml vial of Glycosil (Glycosan BioSystems)
  • 0.5‐ml vial of Extralink (Glycosan BioSystems)
  • DG Water (Glycosan BioSystems)
  • 500 µg/ml commercial (e.g., Sigma) or laboratory‐prepared laminin stock solution (or other sterile, cellular matrix protein in aqueous solution): prepared according to the manufacturer's instructions, if commercially obtained
  • 37°C water bath
  • 1‐ml syringes with long‐tip 20‐G × 1½‐in. needles, sterile
  • 37°C shaking or rocking incubator

Basic Protocol 4: Cell Growth on HA‐Derived Hydrogel Surface

  Materials
  • 7.5‐ml Extracel Hydrogel kit (Glycosan BioSystems)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 1–2 × 104 cells/ml medium suspension of cultured cells of interest: prepared according to standard procedures (e.g., see unit 1.1)
  • Cell culture medium with serum
  • 0.05% trypsin EDTA (VWR)
  • 10× collagenase/hyaluronidase (StemCell Technologies)
  • 37°C water bath
  • 1‐ml syringes with long‐tip 20‐G × 1½‐in. needles, sterile
  • 37°C shaking or rocking incubator
  • 15‐ml sterile, conical tubes
  • 24‐well tissue culture plates
  • Sterile plate‐sealing film (e.g., Axy Seal, VWR) and roller
  • Light microscope (10× magnification)

Basic Protocol 5: Cell Encapsulation in HA‐Derived Hydrogels

  Materials
  • 7.5‐ml Extracel Hydrogel Kit (Glycosan BioSystems) containing:
  • 10× collagenase/hyaluronidase (StemCell Technologies)
  • Sterile phosphate‐buffered saline (PBS)
  • ∼0.4–2 × 104 cells/ml medium suspension of cultured cells of interest: prepared according to standard procedures (e.g., see unit 1.1)
  • Cell culture medium with and without serum
  • 37°C water bath
  • 1‐ml syringes with long‐tip 20‐G × 1½‐in. needles, sterile
  • 37°C shaking or rocking incubator
  • 24‐well plate with tissue culture inserts (e.g., 6.5‐mm Costar tissue culture–treated polycarbonate membrane polystyrene plates, Corning; 8.0‐µm pore size Millicel, Millipore
  • 35‐mm sterile petri dishes
  • Surgical scalpel
  • 15‐ml conical centrifuge tube

Basic Protocol 6: HA‐Derived Hydrogels for Tumor Xenografts

  Materials
  • Extracel Hydrogel kit (Glycosan BioSystems) containing:
    • Glycosil
    • Gelin‐S
    • Extralink
    • DG Water
  • Tumor cells
  • Cell culture medium (without serum)
  • Research animals
  • Iodine and 70% (v/v) ethanol and sterile swabs
  • 37°C water bath
  • 1‐ml syringes with long‐tip 20‐G × 1½‐in. needles, sterile
  • 37°C shaking or rocking incubator
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Figures

Videos

Literature Cited

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