Three‐Dimensional Patterning of the ECM Microenvironment Using Magnetic Nanoparticle Self Assembly

Jiyun Kim1, Kandice Tanner1

1 Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institutes (NIH), Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 25.3
DOI:  10.1002/0471143030.cb2503s70
Online Posting Date:  March, 2016
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This protocol describes a way to introduce topography to three‐dimensional (3D) biomaterials. The self‐assembling behavior of magnetic particles can be exploited to form nanoscale to microscale fibers, such that one can dissect the contribution of topography on cell behavior, which is independent of other physical properties of the biomaterial (e.g., stiffness). The magnetic particles are chemically cross‐linked with several extracellular matrix (ECM) proteins and then using magnetic force‐mediated assembly, one can program aligned nanofibers in a 3D hydrogel. This process allows the creation of diverse topographic patterns in 3D, including isotropic, anisotropic (fibril), or interfaced architectures, without changing the bulk stiffness of the scaffold material. This anisotropic architecture guides the dendritic protrusions of cells, which can be compared to cells grown in an isotropic architecture lacking spatial guidance cues. Several cell types, such as fibroblasts and neurons, have been cultured in this engineered 3D matrix. This technology provides an easy way to construct nano‐bio interfaces for various biomedical engineering applications as well as dissect the role of topography in various cell behaviors. © 2016 by John Wiley & Sons, Inc.

Keywords: three‐dimensional cell culture; extracellular matrix; topography; magnetic particles; magnetic field‐directed self‐assembly; biomaterial; nanocomposite material

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Creating an Anisotropic ECM Microenvironment by Nanoparticle Assembly
  • Support Protocol 1: Chemical Cross‐Linking of the ECM Proteins on the Surface of Magnetic Nanoparticles
  • Support Protocol 2: Staining and Imaging Cells in a 3D Anisotropic Matrix
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Creating an Anisotropic ECM Microenvironment by Nanoparticle Assembly

  • Matrigel (Corning, cat. no. 354230) or hyaluronic acid (e.g., HyStemTM Hydrogel Kit; ESI BIO, cat. no. GS311)
  • Cell culture medium (see recipe)
  • Serum‐free culture medium (see recipe)
  • Cells (e.g., NIH 3T3 fibroblasts) growing in culture
  • ECM protein‐coated superparamagnetic particles ( protocol 2)
  • LabTek 8‐well chambered coverglass (Thermo Scientific, cat. no. 155411) or 12‐well cell culture plate (Corning, cat. no. 3512)
  • 37°C, 10% CO 2 cell culture incubator
  • NdFeB block magnet, 25.4 mm × 25.4 mm × 25.4 mm (K&J Magnetics, Inc., cat. no. B888)
  • Microscope with 10× objective
  • Additional reagents and equipment for mammalian cell tissue culture, trypinization, and counting cells using a hemacytometer (unit 1.1; Phelan and May, )

Support Protocol 1: Chemical Cross‐Linking of the ECM Proteins on the Surface of Magnetic Nanoparticles

  • 300‐nm carboxyl‐superparamagnetic beads (AdemTech, cat. no. 02131)
  • 10× activation buffer (AdemTech, cat. no. 10101)
  • 1‐ethyl‐3‐(3‐dimethylaminopropyl carbodiimide hydrochloride) (EDC; AdemTech, cat. no. 10403)
  • 10× storage buffer (AdemTech, cat. no. 10201)
  • Bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A7906)
  • Fibronectin, labeled with HiLyte Fluor 488 (Cytoskeleton, Inc., cat. no. FNR02)
  • Laminin, labeled with rhodamine (Cytoskeleton, Inc., cat. no. LMN01)
  • 1.5‐ml microcentrifuge tubes
  • Shaker (mixer)

Support Protocol 2: Staining and Imaging Cells in a 3D Anisotropic Matrix

  • Cells grown in an anisotropic 3D matrix (Basic Protocol 1)
  • 4% (w/v) paraformaldehyde (see recipe)
  • Phosphate‐buffered saline, pH 7.2 (PBS; KD Medical, cat. no. RGE‐3190)
  • Blocking buffer (see recipe)
  • Phalloidin, conjugated to Alexa Fluor 633 (Life Technologies, cat. no. A22284)
  • Hoechst 33342 (Life Technologies, cat. no. H3570)
  • Confocal microscope
  • Additional reagents and equipment for immunofluorescence staining (unit 4.1; Donaldson, ) and confocal microscopy (unit 4.5; Smith, )
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Literature Cited

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