Patterning on Topography for Generation of Cell Culture Substrates with Independent Nanoscale Control of Chemical and Topographical Extracellular Matrix Cues

Emily N. Sevcik1, John M. Szymanski1, Quentin Jallerat1, Adam W. Feinberg2

1 Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, 2 Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.23
DOI:  10.1002/cpcb.25
Online Posting Date:  June, 2017
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Abstract

The cell microenvironment plays an important role in many biological processes, including development and disease progression. Key to this is the extracellular matrix (ECM), a complex biopolymer network serving as the primary insoluble signaling network for physical, chemical, and mechanical cues. In vitro, the ability to engineer the ECM at the micro‐ and nanoscales is a critical tool to systematically interrogate the influence of ECM properties on cellular responses. Specifically, both topographical and chemical surface patterning has been shown to direct cell alignment and tissue architecture on biomaterial surfaces, however, it has proven challenging to independently control these surface properties. This protocol describes a method termed Patterning on Topography (PoT) to engineer 2D nanopatterns of ECM proteins onto topographically complex substrates, which enables independent control of physical and chemical surface properties. Applications include interrogation of fundamental cell‐surface interactions and engineering interfaces that can direct cell and/or tissue function. © 2017 by John Wiley & Sons, Inc.

Keywords: extracellular matrix; microtopography; fibronectin; biomaterials; microcontact printing; surface initiated assembly

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Microcontact Printing of ECM Patterns on PIPAAm‐Coated Coverslips
  • Support Protocol 1: Preparation of PDMS Stamps for µCP of ECM Protein Patterns on PIPAAm
  • Support Protocol 2: Preparation of PIPAAm‐Coated Coverslips
  • Support Protocol 3: Labeling of ECM Proteins (LAM, FN, COL IV) with SE‐Conjugated Dyes
  • Basic Protocol 2: Modified SIA Technique for PoT of ECM Proteins
  • Support Protocol 4: Generation of PDMS Substrates with 10‐ to 100‐μm‐Deep Topographic Features for Use as Substrates in PoT
  • Support Protocol 5: Generation of PDMS Topographic Substrates from Rough Materials
  • Support Protocol 6: Cell Seeding on PoT‐Printed Substrates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microcontact Printing of ECM Patterns on PIPAAm‐Coated Coverslips

  Materials
  • PDMS stamps ( protocol 2)
  • Ethanol (200 proof)
  • Distilled water
  • Nitrogen gun or compressed sterile air stream
  • Frozen ECM protein aliquot (FN, COL IV, or LAM; see reciperecipes)
  • Frozen fluorescently labeled ECM protein (optional) [see protocol 4 for succinimidyl ester (SE) labeling of proteins]
  • Sonicator (Branson 3510)
  • 100‐mm diameter petri dishes
  • Micropipettes and micropipette tips
  • Tweezers
  • PIPAAm‐coated coverslips (see protocol 3)
  • Scientific marker

Support Protocol 1: Preparation of PDMS Stamps for µCP of ECM Protein Patterns on PIPAAm

  Materials
  • Chrome mask (Photosciences; acquire in step 2)
  • SPR 220.3 developer (MCF 26A) (Microchem)
  • Distilled water
  • Ethanol (200 proof)
  • SPR 220.3 photoresist (Microchem)
  • Nitrogen or compressed air for drying wafers
  • Sylgard PDMS 184 (see recipe)
  • Computer‐Aided Design (CAD) software
  • Hot plate
  • Rotator
  • Glass rectangular coverslips/wafers (Fisher, cat. no. 12‐543F)
  • Kimwipes
  • UV lamp
  • Adjustable height lab jack with platform
  • Bunsen burner
  • Spin‐coater
  • Transfer pipets
  • Glass piece with soft part around edge
  • Large binder clips
  • Stereomicroscope
  • 150‐mm diameter petri dishes
  • Utility knife
  • Razor blade

Support Protocol 2: Preparation of PIPAAm‐Coated Coverslips

  Materials
  • 25‐mm circular no. 1 coverslips
  • Ethanol (200 proof)
  • 10% (w/v) or 40% (w/v) PIPAAm solution (see recipe)
  • Coverslip rack
  • Sonicator (Branson 3510)
  • Spin‐coater (Specialty coating systems G3P‐8)
  • 150‐mm diameter Petri dish
  • Kimwipes
  • UV light in biosafety cabinet, for cell culture studies
  • Micropipettes and pipette tips
  • Positive displacement pipette (optional)

Support Protocol 3: Labeling of ECM Proteins (LAM, FN, COL IV) with SE‐Conjugated Dyes

  Materials
  • Frozen ECM protein aliquot(s) (FN, COL IV, or LAM; see reciperecipes)
  • SE buffer (see recipe)
  • Fluorophore‐conjugated SE stock (see recipe)
  • Slide‐a‐lyzer dialysis kit (Thermo Fisher, cat. no. PI66332) containing:
    • Dialysis cassettes
    • Needles
    • Syringes
  • Sterile 1× phosphate‐buffered saline (PBS; GE Healthcare Life Sciences, cat. no. SH30256.01)
  • Sterile distilled water
  • 1.5‐ml microcentrifuge tubes
  • 200‐ml beakers

Basic Protocol 2: Modified SIA Technique for PoT of ECM Proteins

  Materials
  • Topographic PDMS substrates (see protocol 6 or 5)
  • Ethanol (200 proof)
  • Distilled water
  • Nitrogen or compressed air
  • PIPAAm‐coated coverslips patterned with ECM protein (from protocol 1)
  • Sonicator (Branson 3510)
  • 35‐mm diameter petri dishes
  • Tweezers
  • 37°C oven
  • Kimwipes
  • Vacuum grease (optional)
  • 12‐well plate
  • 65°C oven

Support Protocol 4: Generation of PDMS Substrates with 10‐ to 100‐μm‐Deep Topographic Features for Use as Substrates in PoT

  Materials
  • Photomask (Photosciences; acquire during step 2)
  • 4‐in. silicon wafers
  • SU‐8 2015 or SU‐8‐2050 photoresist (Microchem)
  • SU‐8 developer (Microchem)
  • Isopropanol
  • Nitrogen or compressed air
  • Trichloro(1H,1H,2H,2H‐perfluorooctyl)silane (Sigma‐Aldrich, cat. no. 448931)
  • Sylgard PDMS 184 (see recipe)
  • Computer‐Aided Design (CAD) software
  • Hot plates
  • Spin‐coater
  • UV lamp
  • Vacuum desiccator
  • 150‐mm petri dishes

Support Protocol 5: Generation of PDMS Topographic Substrates from Rough Materials

  Materials
  • Sylgard PDMS 184 (see recipe)
  • Scissors or utility knife
  • 100‐mm diameter petri dish
  • Paper or sandpaper
  • 65°C oven
  • Razor blade

Support Protocol 6: Cell Seeding on PoT‐Printed Substrates

  Materials
  • PoT‐printed substrates ( protocol 5)
  • 1% Pluronics (see recipe)
  • 1× phosphate‐buffered saline (PBS; GE Healthcare Life Sciences, cat. no. SH30256.01)
  • Cell suspension in medium
  • Biosafety cabinet
  • UV light
  • Hemacytometer
  • Incubator for cell culture
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Figures

Videos

Literature Cited

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