Generation of Micropatterned Substrates Using Micro Photopatterning

Andrew D. Doyle1

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.15
DOI:  10.1002/0471143030.cb1015s45
Online Posting Date:  December, 2009
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Abstract

Micro photopatterning (µPP) has been developed to rapidly test and generate different patterns for extracellular matrix adsorption without being hindered by the process of making physical stamps through nanolithography techniques. It uses two‐photon excitation guided through a point‐scanning confocal microscope to locally photoablate poly(vinyl) alcohol (PVA) thin films in user‐defined computer‐controlled patterns. PVA thin films are ideal for surface blocking, being hydrophilic substrates that deter protein adsorption and cell attachment. Because gold substrates are not used during µPP, all live‐cell fluorescent‐imaging techniques including total internal reflection fluorescence microscopy of GFP–linked proteins can be performed with minimal loss of fluorescence signal. Furthermore, because µPP does not require physical stamps for pattern generation, multiple ECMs or other proteins can be localized within microns of each other. This unit details the setup of µPP. It also provides troubleshooting techniques. Curr. Protoc. Cell Biol. 45:10.15.1‐10.15.35. © 2009 by John Wiley & Sons, Inc.

Keywords: micro photopatterning; micropatterning; extracellular matrix; two‐photon confocal microscopy; photoablation; polyvinyl alcohol; thin film

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Activation of the Glass Surface
  • Alternate Protocol 1: Using Aldehyde‐Terminated Silanes for Surface Activation
  • Basic Protocol 2: Generating Polyvinylalcohol (PVA) Thin Films
  • Basic Protocol 3: Photoablation with Two‐Photon Confocal Microscopy
  • Basic Protocol 4: Dish Quenching
  • Basic Protocol 5: Adsorbing Extracellular Matrix and Plating Cells
  • Support Protocol 1: Setting the Confocal Scan Heads' Rotation Offset
  • Support Protocol 2: Direct Fluorescent Labeling of Fibronectin
  • Support Protocol 3: Using Multiple ECM Proteins with µPP
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Activation of the Glass Surface

  Materials
  • 50% (v/v) nitric acid
  • Deionized or distilled water
  • 200 mM NaOH solution (see recipe)
  • (3‐aminopropyl)trimethoxysilane (APTMS: 97% or higher; Gelest)
  • 50% (v/v) glutaraldehyde (Electron Microscopy Sciences)
  • Aldex (Waste and Compliance Management)
  • Drierite (W.A. Hammond Drierite Company)
  • R‐3603 Tygon tubing (Norton Performance Plastics)
  • 1000‐µl barrier filter pipet tips
  • Low‐pressure air jet
  • Fume hood
  • Thirty MatTek glass‐bottomed dishes (P35G‐1.5‐10‐CMatTek)
  • Carrying tray
  • Pasteur pipets
  • Automatic pipettor
  • 2000‐ml beaker
  • Scale
  • Scintillation vials or other small glass containers
  • Drying oven
  • 500‐ml screw‐top container (e.g., Nalgene) or other similar desiccated containers for storage

Alternate Protocol 1: Using Aldehyde‐Terminated Silanes for Surface Activation

  • Absolute ethanol (200 proof)
  • Triethoxysilylbutraldehyde (TESBA) or 11‐(Triethoxysilyl)undecanal (TESUDA; both from Gelest)

Basic Protocol 2: Generating Polyvinylalcohol (PVA) Thin Films

  Materials
  • Distilled water
  • Poly(vinyl) alcohol powder (mol. wt. between 13,000 and 100,000; 98% hydrolyzed minimum; Sigma)
  • 2 N HCl
  • 5 M NaCl
  • 400‐ml glass beaker
  • Stirrer/hot plate
  • 50‐ml conical tubes
  • Scale
  • Flea Micro magnetic stir bar (VWR)
  • 50‐ml Steriflip (0.2‐µm pore size)
  • Gel vacuum pump
  • High‐pressure compressed air source (e.g., compressed air cylinder)
  • Vortex
  • 5 to 10 MatTek dishes with activated glass surface ( protocol 1 or protocol 2)
  • Pipettor
  • Spincoater with a chuck capable of accepting 50‐mm or smaller items (A WS‐400B‐6NPP/LITE spincoater from Laurell Technologies shown in Fig. is used here)
  • Scintillation vial or 35‐mm tissue culture dish
  • Nalgene 500‐ml screw top or other similar containers for storage

Basic Protocol 3: Photoablation with Two‐Photon Confocal Microscopy

  Materials
  • Glass cleaner
  • Immersion oil
  • Zeiss 510 LSM NLO confocal microscope or later model with 1.5‐W minimum tunable two‐photon titanium:Sapphire laser, and a 633‐nm HeNe2 laser (5 mW power output)
  • AIM software (Zeiss MicroImaging)
  • 63× oil immersion objective with numerical aperture of 1.3 or higher capable of NLO transmission
  • PVA thin film‐coated MatTek dishes ( protocol 3)

Basic Protocol 4: Dish Quenching

  Materials
  • µPP‐patterned dishes (Basic Protocols protocol 11 through protocol 43)
  • 200 mM ethanolamine buffer (see recipe)
  • Sodium borohydride solution (NaBH 4; see recipe)
  • 1 M NaOH solution (see recipe)
  • Phosphate‐buffered saline (PBS; Hyclone, cat. no. SH30264.02)
  • Phosphate‐buffered saline (PBS) with penicillin/streptomycin and fungizone (see recipe)
  • Storage containers
  • Scale
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 5: Adsorbing Extracellular Matrix and Plating Cells

  Materials
  • Fibronectin at 2 mg/ml concentration in PBS or other suitable buffer
  • Phosphate‐buffered saline (PBS) with 0.1% (v/v) pluronic F‐127 (see recipe)
  • µPP patterned dishes (Basic Protocols protocol 11 through protocol 54)
  • Lyophilized bovine serum albumin (BSA)
  • Phosphate‐buffered saline (PBS; Hyclone, cat. no. SH30264.02)
  • 2 M NaCl solution
  • Phosphate‐buffered saline (PBS) with penicillin/streptomycin and fungizone (see recipe)
  • NIH/3T3 cells (ATCC) grown to 60% to 70% confluency in a 100‐mm diameter dish in 10% CO 2 incubator
  • Hanks balanced salt solution (HBSS; Invitrogen)
  • 0.5% (w/v) trypsin/EDTA solution (Invitrogen)
  • NIH/3T3 fibroblast culture medium (see recipe)
  • Tissue culture hood
  • 37°C waterbath
  • 400‐ml beaker
  • Stirrer/hot plate
  • Scale
  • 50‐ml conical tubes
  • Glass test tube capable of holding 30 to 50 ml
  • Flea Micro magnetic stir bar (VWR)
  • Digital thermometer
  • Ice in an ice bucket
  • Vacuum aspirator
  • Airtight storage container
  • Benchtop swinging‐bucket rotor centrifuge with adapters for 50‐ml conical tubes
  • Inverted microscope equipped with a 10× phase contrast objective

Support Protocol 1: Setting the Confocal Scan Heads' Rotation Offset

  Materials
  • Zeiss 510 LSM NLO confocal microscope or later model with 1.5 W minimum tunable two‐photon titanium:sapphire laser, and a 633‐nm HeNe2 laser (5‐mW power output), and a 543‐nm HeNe1 laser (1‐mW power output)
  • AIM software (Zeiss MicroImaging)
  • PVA thin film–coated MatTek dishes ( protocol 3; for option 1)
  • Arc lamp
  • Grid slide, Objektträger (for option 2; Zeiss, cat. no. 474028)
  • Fluorescent highlighter, any color (for option 2)
  • Kimwipes

Support Protocol 2: Direct Fluorescent Labeling of Fibronectin

  Materials
  • NHS‐ester‐based fluorescent dye of choice (several are available from Invitrogen and Pierce)
  • Dimethyl sulfoxide (DMSO)
  • 500 to 1000 µl of fibronectin at 2 mg/ml concentration or 2 mg of lyophilized fibronectin
  • 100 mM borate buffer, pH 9.0 (see recipe)
  • Slide‐A‐Lyzer (Pierce)
  • 1.5‐ml microcentrifuge tubes
  • Aluminum foil
  • End‐over‐end rotating mixer, e.g., Labquake rotating mixer (sometimes termed a rotisserie shaker)
  • Desalting spin column or dye‐removal columns capable of ∼1 ml volumes (Pierce)
  • Centrifuge capable of 10,000 × g with 15‐ml conical tube holders

Support Protocol 3: Using Multiple ECM Proteins with µPP

  • Two different, fluorescently labeled ECM molecules/growth factors at the proper final concentration (user defined)
  • 1% (w/v) heat‐denatured BSA solution (prepare fresh and keep <1 day)
  • Phosphate‐buffered saline (PBS) with 0.1% pluronic F‐127 (see recipe)
  • PVA thin film–coated MatTek dishes ( protocol 3)
  • Permanent marker
  • AIM software (Zeiss MicroImaging)
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Figures

Videos

Literature Cited

Literature Cited
   Chen, S., Kancharla, V.V., and Lu, Y. 2003. Laser‐based microscale patterning of biodegradable polymers for biomedical applications. Int. J. of Material & Product Technol. 18:457‐468.
   Doyle, A.D., Wang, F.W., Matsumoto, K., and Yamada, K.M. 2009. One‐dimensional topography underlies three‐dimensional fibrillar cell migration. J. Cell. Biol. 184:481‐490.
   Du, J.Z., Sun, T.M., Weng, S.Q., Chen, X.S., and Wang, J. 2007. Synthesis and characterization of photo‐cross‐linked hydrogels based on biodegradable polyphosphoesters and poly(ethylene glycol) copolymers. Biomacromolecules 8:3375‐3381.
   Hermanson, G.T. 1996. Bioconjugate Techniques. 1st ed. Academic Press, San Diego, Calif.
   Lehnert, D., Wehrle‐Haller, B., David, C., Weiland, U., Ballestrem, C., Imhof, B., and Bastmeyer, M. 2004. Cell behavior on micropatterned substrata: Limits of extracellular matrix geometry for spreading and adhesion. J. Cell Sci. 117:41‐52.
   Matsumoto, M., Imai, K., and Kazusa, Y. 1958. Ultraviolet spectra of polyvinyl alcohol. J. Polymer Sci. 117:426‐428.
   Mrksich, M., Chen, C.S., Xia, Y., Dike, L.E., Ingber, D.E., and Whitesides, G.M. 1996. Controlling cell attachment on contoured surfaces with self‐assembled monolayers of alkanethiolates on gold. Proc. Natl. Acad. Sci. U.S.A. 93:10775‐10778.
   Peppas, N.A. and Merrill, E.W. 1977. Development of semicrystalline poly(vinyl alcohol) hydrogels for biomedical applications. J. Biomed. Mater. Res. 11:423‐434.
   Singhvi, R., Kumar, A., Lopez, G.P., Stephanopoulos, G.N., Wang, D.I., Whitesides, G.M., and Ingber, D.E. 1994. Engineering cell shape and function. Science 264:696‐698.
   Yamato, M., Konno, C., Koike, S., Isoi, Y., Shimizu, T., Kikuchi, A., Makino, K., and Okano, T. 2003. Nanofabrication for micropatterned cell arrays by combining electron beam‐irradiated polymer grafting and localized laser ablation. J. Biomed. Mater. Res. A 67:1065‐1075.
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