Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering

Wan‐Ju Li1, Rocky S. Tuan2

1 University of Wisconsin‐Madison, Madison, Wisconsin, 2 National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 25.2
DOI:  10.1002/0471143030.cb2502s42
Online Posting Date:  March, 2009
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Nanofibers fabricated by electrospinning are morphological mimics of fibrous components of the native extracellular matrix, making nanofibrous scaffolds ideal for three‐dimensional cell culture and tissue engineering applications. Although electrospinning is not a conventional technique in cell biology, the experimental setup may be constructed in a relatively straightforward manner, and the procedure can be carried out by individuals with limited engineering experience. Here, we detail a protocol for electrospinning of nanofibers and provide relevant specific details concerning the optimization of fiber formation (Basic Protocol 1). The protocol also includes conditions required for preparing biodegradable polymer solutions for the fabrication of nonwoven and aligned nanofibrous scaffolds suitable for various cell/tissue applications. In addition, information on effective cell loading into nanofibrous scaffolds and cellular constructs grown in a bioreactor is provided (Basic Protocol 2). Instructions for building the electrospinning apparatus are also included (see the Support Protocol). Curr. Protoc. Cell Biol. 42:25.2.1‐25.2.12. © 2009 by John Wiley & Sons, Inc.

Keywords: nanofiber; electrospinning; scaffold; tissue engineering; three‐dimensional culture

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Fabrication of Nanofibrous Scaffolds
  • Basic Protocol 2: Nanofibrous Scaffolds for Cell Culturing
  • Support Protocol 1: Construction of an Electrospinning Apparatus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Fabrication of Nanofibrous Scaffolds

  • PLLA (mol. wt. 50,000; Polysciences)
  • N,N‐Dimethyformamide (DMF; Fisher Scientific)
  • Chloroform (Fisher Scientific)
  • PCL (mol. wt. 80,000; Aldrich)
  • Tetrahydrofuran (THF; Fisher Scientific)
  • Glass threaded vials
  • Analytical balance
  • Parafilm
  • Vortex mixer
  • Aluminum foil
  • Electrospinning apparatus (see protocol 3)
  • Glass slides
  • Glass coverslips
  • 10‐ml glass syringe fitted with an 18‐G blunt needle
  • Sharp surgical blade
  • Vacuum desiccator

Basic Protocol 2: Nanofibrous Scaffolds for Cell Culturing

  • Graded ethanol series [70% (v/v), 50% (v/v), and 25% (v/v) ethanol, see recipe]
  • Sterile distilled water
  • Hank's Balanced Salt Solution (HBSS), sterile
  • Cell suspension in 10% serum‐containing culture medium (cell density = 20 × 106/ml; see recipe for culture medium)
  • Serum‐containing culture medium (see recipe)
  • Surgical blade
  • Nanofibrous sheet/scaffolds (see protocol 1)
  • Petri dish
  • UV light source
  • Bibulous filter paper (Whatman 3 MM)
  • Pipettor
  • Multi‐well culture plate
  • Tissue culture incubator
  • Rotary wall vessel (RWV) bioreactor (Synthecon)

Support Protocol 1: Construction of an Electrospinning Apparatus

  • High‐density polyethylene (HDPE) plate
  • Polycarbonate plate
  • HDPE square rods
  • Wooden plate (20‐in. × 20‐in.)
  • Copper plate (10‐in. × 10‐in.)
  • Teflon plate (20‐in. × 20‐in.)
  • Glass syringe
  • 18‐G blunt metal needle
  • High‐voltage DC power supply
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Literature Cited

Literature Cited
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