Paper‐based Invasion Assays for Quantifying Cellular Movement in Three‐dimensional Tissue‐like Structures

C. Chad Lloyd1, Matthew W. Boyce1, Matthew R. Lockett2

1 Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, 2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.22
Online Posting Date:  June, 2017
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Abstract

To elucidate the chemical and environmental conditions that promote invasion of cancer cells, an assay is needed in which the chemical landscape of a tumor‐like environment can be experimentally manipulated and probed. The three‐dimensional paper‐based invasion assays described here simulate poorly vascularized tissue and allow the invasion of cancerous cells to be visualized and quantified. These cultures are easy to assemble and allow multiple invasion assays to be performed in parallel. By using different materials to control gradients formed across the culture, the chemotactic potential of small molecules can be evaluated in a more representative tissue microenvironment. © 2017 by John Wiley & Sons, Inc.

Keywords: chemotaxis; hypoxia; invasion; oxygen gradients; tumor microenvironment

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assembling an Invasion Stack Assay with an Oxygen Gradient
  • Basic Protocol 2: Quantification and Analysis of Cell Invasion with Fluorescence Imaging
  • Support Protocol 1: Preparing Paper Scaffolds
  • Support Protocol 2: Preparing Transparency Films
  • Support Protocol 3: Preparing PDMS Films
  • Support Protocol 4: Fabricating Custom Acrylic Holders
  • Support Protocol 5: Making a Stacking Apparatus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Assembling an Invasion Stack Assay with an Oxygen Gradient

  Materials
  • Cell line of choice (e.g., MDA‐MB‐231 cells, ATCC)
  • Appropriate culture medium for cells (e.g., RPMI 1640 with antibiotics, see recipe)
  • 70% (v/v) ethanol
  • 1× Dulbecco's phosphate‐buffered saline (DPBS), no calcium or magnesium (Gibco)
  • TrypLE Express (Thermo Fisher Scientific)
  • Matrigel (9.4 mg/ml, growth factor‐reduced, Corning, keep on ice)
  • Trypan blue
  • 10‐cm cell culture dish
  • Cell culture hood
  • Incubator (37°C, 5% CO 2)
  • Pasteur pipets
  • Sterilized paper scaffolds (see protocol 3)
  • 15‐ml Falcon tube
  • 1.5‐ml microcentrifuge tubes
  • Sterile 6‐ and 12‐well culture plates
  • 10‐ml serological pipet and automatic pipet
  • Micropipets and tips
  • Sterilized tweezers
  • Bright‐field microscope
  • Hemocytometer
  • Hot plate
  • Sterilized transparencies, closed and open (see protocol 4)
  • Sterilized PDMS film (see protocol 5)
  • Sterilized acrylic holder (see protocol 6)
  • Stacking apparatus (optional; see protocol 7)
  • Sterilized Phillips‐head screwdriver
  • Sterilized 4‐40 5/8‐in. flat‐head Phillips screws (four)

Basic Protocol 2: Quantification and Analysis of Cell Invasion with Fluorescence Imaging

  Materials
  • 1× PBS (see recipe)
  • Invasion stack (see protocol 1)
  • 10 mM FDA (see recipe)
  • 12‐well plate
  • Phillips‐head screwdriver
  • Tweezers
  • 15‐ml Falcon tube
  • Timer
  • Incubator (37°C, 5% CO 2)
  • Laser scanning fluorescence scanner (e.g., Typhoon 9400, GE Healthcare Life Sciences)
  • Imaging software (e.g., ImageJ, FIJI, MatLab)
NOTE: Disassembly and analysis can be done in a non‐sterile environment.

Support Protocol 1: Preparing Paper Scaffolds

  Materials
  • 70% (v/v) ethanol
  • Vector‐based drawing software (e.g., Adobe Illustrator or CorelDraw)
  • Whatman lens cleaning tissue 105, 200 × 300 mm (Sigma‐Aldrich)
  • Transparency sheets, 8.5 × 11 in. (e.g., from office supply store)
  • Single‐ and double‐sided tape
  • Solid ink printer (Xerox ColorQube 8570 or other printer using wax‐based inks)
  • Utility knife
  • Drying oven
  • Pyrex baking dish
  • Surgical scissors
  • 1‐mm tissue punch
  • Transilluminator table (e.g., Foto/WL, Fotodyne)
  • Biological safety cabinet, equipped with a UV sterilization light

Support Protocol 2: Preparing Transparency Films

  Materials
  • 70% (v/v) ethanol
  • Vector‐based drawing software (e.g., Adobe Illustrator or CorelDraw)
  • Transparency sheets, 8.5 × 11 in. (e.g., from office supply store)
  • Craft cutter, with associated cutting mat (e.g., Silver Bullet Cutter)
  • Tape
  • Autoclave

Support Protocol 3: Preparing PDMS Films

  Materials
  • 1× PBS (see recipe) with 0.3% Triton X‐100
  • Sylgard 184 silicon elastomer and curing agent
  • 70% (v/v) ethanol
  • Two glass or acrylic plates with flat surfaces (¼‐in. thick acrylic, 6 × 6 in.)
  • 50‐ml Falcon tube
  • Vacuum oven or desiccator (optional)
  • Transparency sheet (e.g., from office supply store)
  • Binder clips
  • Utility knife
  • 1‐mm tissue punch
  • Biological safety cabinet equipped with a UV sterilization light
  • Profilometer (KLA‐Tencor P‐6; optional)
  • Spin coater

Support Protocol 4: Fabricating Custom Acrylic Holders

  Materials
  • 70% (v/v) ethanol (optional)
  • Vector‐based drawing software (e.g., Adobe Illustrator or CorelDraw)
  • Laser cutter (e.g., ILS12.75, Universal Laser Systems)
  • 6 × 6–in. sheet of ¼‐in. transparent cast acrylic (McMaster Carr)
  • Drill with 1/8‐in. drill bit
  • 4‐40 tap
  • Tap wrench
  • Biological safety cabinet with UV light (optional)
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Figures

Videos

Literature Cited

Literature Cited
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