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Series Editors
Leslie Wilson
Department of Molecular, Cellular and Developmental Biology
University of California
Santa Barbara, California
Phong Tran
Department of Cell and Developmental Biology
University of Pennsylvania
Philadelphia, Pennsylvania

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Contributors
Muhammad Ali
INSERM U1026, and University of Bordeaux Segalen, Bordeaux, France
Buzz Baum
Medical Research Council – Laboratory for Molecular Cell Biology, University
College London, London, United Kingdom
Jonathan M. Be´lisle
Centre de Recherche de l’Hoˆpital Maisonneuve-Rosemont, and De´partement
d’Ophtalmologie et Institut de Ge´nie Biome´dical, Universite´de Montre´al,
Montre´al, Que´bec, Canada
Keith A. Brown
Department of Chemistry, and International Institute for Nanotechnology,
Northwestern University, Evanston, Illinois, USA
O¨zgu¨l Demir Bulut
Institute for Biological Interfaces, Karlsruhe Institute of Technology (KIT),
Karlsruhe, Germany
Maria D. Cabezas
Department of Chemistry, Northwestern University, Evanston, Illinois, USA
E. Ada Cavalcanti-Adam
Department of New Materials and Biosystems, Max Planck Institute for Intelligent
Systems, Stuttgart, and Department of Biophysical Chemistry, Institute for
Physical Chemistry, University of Heidelberg, Heidelberg, Germany
Arnold Chen
Micro-Nano Innovations (MiNI) Laboratory, Department of Biomedical
Engineering, University of California, Davis, California, USA
Christopher S. Chen
Department of Bioengineering, University of Pennsylvania, Philadelphia,
Pennsylvania USA; Department of Biomedical Engineering, Boston University,
and Wyss Institute for Biologically Inspired Engineering, Harvard University,
Boston, Massachusetts, USA
Guoping Chen
Tissue Regeneration Materials Unit, International Center for Materials
Nanoarchitectonics, National Institute for Materials Science, Tsukuba, Ibaraki,
Japan
Jong-Cheol Choi
Department of Mechanical Engineering, Pohang University of Science and
Technology, Pohang, Gyeongbuk, South Korea
Manuela Medina Correa
INSERM U1026, and University of Bordeaux Segalen, Bordeaux, France
xi

Santiago Costantino
Centre de Recherche de l’Hoˆpital Maisonneuve-Rosemont, and De´partement
d’Ophtalmologie et Institut de Ge´nie Biome´dical, Universite´de Montre´al,
Montre´al, Que´bec, Canada
Cle´ment V.M. Cremmel
Laboratory for Surface Science and Technology, Department of Materials, ETH
Zurich, Zurich, Switzerland
Matthew J. Dalby
Centre for Cell Engineering, Institute of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow, United Kingdom
Ravi A. Desai
Department of Bioengineering, University of Pennsylvania, Philadelphia,
Pennsylvania USA; Max Planck Institute for Molecular Cell Biology and Genetics,
Dresden, Germany; Medical Research Council, National Institute of Medical
Research, and University College London, London, United Kingdom
Raphae¨l Devillard
INSERM U1026, and University of Bordeaux Segalen, Bordeaux, France
Junsang Doh
School of Interdisciplinary Bioscience and Bioengineering (I-Bio), and
Department of Mechanical Engineering, Pohang University of Science and
Technology, Pohang, Gyeongbuk, South Korea
Daniel J. Eichelsdoerfer
Department of Chemistry, Northwestern University, Evanston, Illinois, USA
Katarzyna M. Gadomska
Department of New Materials and Biosystems, Max Planck Institute for Intelligent
Systems, Stuttgart, and Department of Biophysical Chemistry, Institute for
Physical Chemistry, University of Heidelberg, Heidelberg, Germany
Andre´s J. Garcı´a
Woodruff School of Mechanical Engineering, Georgia Institute of Technology, and
Petit Institute for Bioengineering and Bioscience, Atlanta, Georgia, USA
Jose´R. Garcı´a
Woodruff School of Mechanical Engineering, Georgia Institute of Technology, and
Petit Institute for Bioengineering and Bioscience, Atlanta, Georgia, USA
Fabien Guillemot
INSERM U1026, and University of Bordeaux Segalen, Bordeaux, France
Bertrand Guillotin
INSERM U1026, and University of Bordeaux Segalen, Bordeaux, France
Rebecca P. Huber
Laboratory for Surface Science and Technology, Department of Materials, ETH
Zurich, Zurich, Switzerland
xii Contributors

Kazuyoshi Itoga
Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s
Medical University, Tokyo, Japan
Jan-Willi Janiesch
Department of New Materials and Biosystems, Max Planck Institute for Intelligent
Systems, Stuttgart, and Department of Biophysical Chemistry, Institute for
Physical Chemistry, University of Heidelberg, Heidelberg, Germany
Hong-Ryul Jung
School of Interdisciplinary Bioscience and Bioengineering (I-Bio), Pohang
University of Science and Technology, Pohang, Gyeongbuk, South Korea
Je´roˆme Kalisky
INSERM U1026, and University of Bordeaux Segalen, Bordeaux, France
Jiwoo Kang
School of Interdisciplinary Bioscience and Bioengineering (I-Bio), Pohang
University of Science and Technology, Pohang, Gyeongbuk, South Korea
Miju Kim
School of Interdisciplinary Bioscience and Bioengineering (I-Bio), Pohang
University of Science and Technology, Pohang, Gyeongbuk, South Korea
Jun Kobayashi
Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s
Medical University, Tokyo, Japan
Virginie Ke´riquel
INSERM U1026, and University of Bordeaux Segalen, Bordeaux, France
Ilia Louban
Department of New Materials and Biosystems, Max Planck Institute for Intelligent
Systems, Stuttgart, and Department of Biophysical Chemistry, Institute for
Physical Chemistry, University of Heidelberg, Heidelberg, Germany
Javier Mazzaferri
Centre de Recherche de l’Hoˆpital Maisonneuve-Rosemont, Montre´al, Que´bec,
Canada
Rachel McKendry
London Centre for Nanotechnology and Department of Medicine, University
College London, London, United Kingdom
Laura E. McNamara
Centre for Cell Engineering, Institute of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow, United Kingdom
Chad A. Mirkin
Department of Chemistry; International Institute for Nanotechnology, and
Department of Materials Science and Engineering, Northwestern University,
Evanston, Illinois, USA
xiiiContributors

Milan Mrksich
Department of Chemistry; International Institute for Nanotechnology, and
Department of Biomedical Engineering, Northwestern University, Evanston,
Illinois, USA
Teruo Okano
Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s
Medical University, Tokyo, Japan
Emeline Page`s
INSERM U1026, and University of Bordeaux Segalen, Bordeaux, France
Tingrui Pan
Micro-Nano Innovations (MiNI) Laboratory, Department of Biomedical
Engineering, University of California, Davis, California, USA
Remigio Picone
Department of Cell Biology, Harvard Medical School; Department of Pediatric
Oncology, Howard Hughes Medical Institute; Department of Pediatric Oncology,
Dana-Farber Cancer Institute, Boston, Massachusetts, USA; London Centre for
Nanotechnology and Department of Medicine; Medical Research Council –
Laboratory for Molecular Cell Biology, and CoMPLEX, University College London,
London, United Kingdom
Ilia Platzman
Department of New Materials and Biosystems, Max Planck Institute for Intelligent
Systems, Stuttgart, and Department of Biophysical Chemistry, Institute for
Physical Chemistry, University of Heidelberg, Heidelberg, Germany
Alexander Revzin
Department of Biomedical Engineering, University of California, Davis, California,
USA
Murielle Re´my
INSERM U1026, and University of Bordeaux Segalen, Bordeaux, France
Natalia M. Rodriguez
Department of Bioengineering, University of Pennsylvania, Philadelphia,
Pennsylvania; Department of Biomedical Engineering, Boston University, and
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston,
Massachusetts, USA
Ankur Singh
Woodruff School of Mechanical Engineering, Georgia Institute of Technology, and
Petit Institute for Bioengineering and Bioscience, Atlanta, Georgia, USA
John H. Slater
Department of Biomedical Engineering, Duke University, Durham, North
Carolina, USA
xiv Contributors

Joachim P. Spatz
Department of New Materials and Biosystems, Max Planck Institute for Intelligent
Systems, Stuttgart, and Department of Biophysical Chemistry, Institute for
Physical Chemistry, University of Heidelberg, Heidelberg, Germany
Nicholas D. Spencer
Laboratory for Surface Science and Technology, Department of Materials, ETH
Zurich, Zurich, Switzerland
Monica P. Tsimbouri
Centre for Cell Engineering, Institute of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow, United Kingdom
Nagaiyanallur V. Venkataraman
Laboratory for Surface Science and Technology, Department of Materials, ETH
Zurich, Zurich, Switzerland
Simone Weigel
Institute for Biological Interfaces, Karlsruhe Institute of Technology (KIT),
Karlsruhe, Germany
Alexander Welle
Institute of Functional Interfaces, Institute for Biological Interfaces, Karlsruhe
Institute of Technology (KIT), Karlsruhe, Germany
Jennifer L. West
Department of Biomedical Engineering, Duke University, Durham, North
Carolina, USA
Siyuan Xing
Micro-Nano Innovations (MiNI) Laboratory, Department of Biomedical
Engineering, University of California, Davis, California, USA
Masayuki Yamato
Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s
Medical University, Tokyo, Japan
Siwei Zhao
Micro-Nano Innovations (MiNI) Laboratory, Department of Biomedical
Engineering, University of California, Davis, California, USA
Christian Zink
Laboratory for Surface Science and Technology, Department of Materials, ETH
Zurich, Zurich, and Phonak AG, Sta¨fa, Switzerland
xvContributors

Preface
Micropatterning refers generally to techniques which provide an experimental con-
trol over the chemical, physical, or geometrical properties of materials at the micron
or submicron scale, and are thus used to produce spatial patterns of these properties.
These techniques, which were often originally designed for application in microelec-
tronics, have spread over most areas of science, including biology. They have proved
particularly useful for cell biology, bridging the gap between the Petri-dish and
complex 3D assays and tissues. At the level of single cells, many environmental
parameters are entangled and assessing their individual contribution to cell physiology
and behavior is often difficult. Micropatterned cell-culture substrates allow to spe-
cifically design tools to quantitatively control the cell microenvironmentin vitroand
to assess the effect of individual parameters, with devices which are almost as easy to
handle as a regular Petri dish. Historically, printing of cell adhesion molecules, such
as collagen or fibronectin, have allowed producing cell culture substrates on which
cells have a well-defined shape and adhesion geometry. Such substrates have been
crucial to demonstrate the role of cell shape, cell spreading area and of geometrical
parameters of cell adhesion on cell survival, proliferation, differentiation, and polar-
ity. The fabrication of micropatterned substrates initially required special expertise
in surface chemistry and sophisticated devices, but their success lead to the devel-
opment of much simpler methods accessible to almost any regular biology lab
(seeChapters 1–6for printing of proteins on various types of substrates, including
printing of multiple proteins and of gradients). Efforts have also been made to make
the process cheaper and more versatile (see maskless techniques inChapters 7–11).
Micropatterning now covers a large number of cell biology applications, from stem
cell culture and differentiation (see e.g.,Chapters 2 and 13) to printing of purified
proteins or other biomolecules forin vitroassays (seeChapters 15 and 1–4of
vol. 120). Moreover, the size of the features which can be printed is now down to
tens of nanometers (seeChapters 12–14). Current micropatterning techniques have
developed further to implement the quantitative control of other aspects of the cell
microenvironment such as 3D geometry (seeChapters 7–15of vol. 121) and mechan-
ical properties (seeChapters 16of vol. 120,3 and 6of vol. 121). Importantly, some of
these tools do not only allow building microcontrolled environments for cultured
cells, but are also measurement tools, giving access to crucial parameters such as
forces (seeChapters 13of vol. 120 and1, 2, 4of vol. 121). Although the technical
basis for most micropatterning methods is very generic, clever variations and adap-
tation are enough to produce tools for very specific applications, such as the study of
collective cell behavior (seeChapter 15of vol. 120), imaging of yeast cells from the
tip (Chapter 14of vol. 120), or local application of forces on individual cells
(Chapter 12of vol. 120). The latest evolutions of micropatterning are meant to
implement temporal control of the micropatterned features (seeChapters 5–11of
vol. 120), to reach full spatio-temporal control of the cell microenvironment.
Matthieu Piel and Manuel The´ry
xvii

Biography
A Tribute to Co-Editor Paul Matsudaira
After more than 20 years a co-Editor of Methods in Cell Biology, Paul Matsudaira
is stepping down. Paul joined the series in 1991 and has been highly instrumental in
the continuation and, importantly, the successful expansion of the Methods series
between then and now. In the earliest days of Methods in Cell Biology, which began
in 1964, founding editor David Prescott (University of Colorado, Boulder), oversaw
publication of the first 20 volumes. Those were also the formative years of modern
cell biology and initially David published only one volume every two years, then one
volume a year, and eventually 2-3 volumes during the last few years that he was
editor. The structure of the series and the breadth of cell biology then was quite dif-
ferent than now, in that David not only was the editor of the series, but because cell
biology was still a small discipline, he could organize each volume by himself. David
stepped down as series editor in 1978 and oversight of the series was assumed by the
American Society for Cell Biology through an Advisory Board, chaired by Keith R.
Porter. The ASCB Advisory Board Committee guided publication of 6 volumes
through Volume 26 with each volume organized by a different editor; a new model
for the series. Still the series output remained relatively low at2 volumes per year.
I was appointed editor by the ASCB Advisory Committee in 1986, and continued as
the sole editor until 1991. At that time it became clear that the field of cell biology
and the methodology for studying cells and their functions were expanding at a gal-
loping pace, so Paul Matsudaira joined me as a co-editor in 1991 to foster develop-
ment and expansion of the series so as to keep pace with the expansion of cell
biology. The expansion of Methods in Cell Biology and our efforts to keep ahead
of new developments in cell biology were extremely successful, thanks in large part
to Paul’s insights, interest in and knowledge of cutting end methods and emerging
disciplines within cell biology. Close to 120 volumes have been published in the
Methods in Cell Biology series with the rate of publication now at 6 volumes per
year, the recent addition of another co-editor, Phong Tran, and a rich pipeline of
future volumes. Early on Paul and I implemented a unique program of theme-focused
methods that has proven highly valuable to the cell biology research community,
which has included groups of volumes such as volum groups focusing on model
organisms in cell biology and on all aspects of microscopy. The theme volumes
approach is unique to Methods in Cell Biology and is a mainstay of the series. It helps
the series stand out as the place where students and researchers can go to learn, for
example, what is the best organism to use to study a specific question, or what is the
best kind of microscopy to use for a particular question and how to do it. Specifically
with Paul’s input, Methods in Cell Biology has expanded greatly keeping pace
with developments in cell biology, with modern methods focusing on the more bio-
physical and quantitative aspects of cell biology, including, just to mention a few,
volumes on atomic force microscopy, cell mechanics, laser tweezers, and computa-
tional methods.
xix

Throughout his career, Paul has been and continues to be a highly innovative ex-
perimentalist and teacher. Paul started his research career in cell biology while an
undergraduate student working for cell biologist and pioneering electron microsco-
pist Thomas Schroeder at the University of Washington Friday Harbor Laboratrories,
which is where I first met him. He obtained his PhD degree at Dartmouth College
with cell biologist David Burgess. in 1981 he became a postdoctoral fellow with
Klaus Weber at the Max Planck Institute for Biophysical Chemistry in Goettingen,
Germany, and subsequently took on a second postdoc with Alan Weeds at the MRC
Laboratory of Molecular Biology in the UK, before joining the faculty of MIT. He
was Professor of Biology and Biological Engineering at the Whitehead Institute at
MIT until 1999 and during his tenure at the Whitehead and MIT he initiated and
directed a number of innovative teaching and research programs. In 1999, he joined
the National University of Singapore (NUS) as Professor of Biological Sciences and
Head of the Department of Biological Sciences and launched the Centre for Bio-
Imaging Sciences. Paul has had a long-standing interest in advancing cell biology
through development of novel methods. For example, he published the book “A
Practical Guide to Protein and Peptide Purification for Micro Sequencing
(Academic Press/Elsevier) in 1993 when the methodology for micro sequencing
of proteins was in its infancy. It is entirely appropriate for me to say that Paul has
always been ahead of the curve in pioneering the newest methods for advancing re-
search and knowledge in cell biology. Paul is also well known as an accomplished
teacher. Paul is not leaving science but rather is continuing to expand his leadership
role in cell biology and biophysics at NUS. I have greatly enjoyed working together
with Paul on the Methods in Cell Biology Series during these many years, and con-
sider him a close friend. I wish him well in his future endeavors and can say easily
that I will miss working with him on the Methods series, but that I still look forward
to many fine dinners with excellent food, wine and of course, our families.
Leslie Wilson
Santa Barbara, CA
xx Biography

I was asked by Les Wilson to assist him as Co-Editor of Methods in Cell Biology,
effectively stepping into the shoes of Paul Matsudaira, who had held this position for
more than twenty years.
At a dinner at the ASCB 2012 meeting, I met with both Paul and Les, and Elsevier
publishers Zoe Kruze and Lisa Tickner, to discuss future directions for MCB.
I learned a lot from these seasoned individuals, and in particular from Paul concern-
ing the anticipation of important methodologies.
I look forward to working with the MCB team. Indeed, Paul has left quite large
shoes to fill. I wish Paul well in his future endeavors.
Phong Tran
Paris
xxiBiography

It has been my pleasure to co-edit Methods in Cell Biology and with Les Wilson
for the past twenty-three years. The editorship began in 1991 with volume 34,
Vectorial Transport of Proteins into and across Membranes, edited by Alan Tartakoff.
Since then we have nurtured and developed within MCB several subseries of volumes
on microscopy, biophysical methods, model cells/organelles/tissues/organisms, and
model processes. Many volumes are classics not only in cell biology but also in
developmental biology and biophysics. We thought that the molecular biology
and biochemical problems studied in the 80’s would become cell biological problems
in the 90’s and in the following decades, that biophysical tools would be needed to
study cells and tissues. Looking back at the past eighty-five volumes, I have a deep
sense of satisfaction that MCB volumes have arrived at the right place, at the
right time.
Les and I have been a team since 1975 when I was a technician for Tom Schroeder
(volume 27, Echinoderm Gametes and Embryos, 1986) at the University of Wash-
ington Friday Harbor Labs. Les would spend the summer at Friday Harbor along with
Joe Bryan, Jim Spudich, Dave Burgess, Ellis Ridgeway, and Ray Rappaport. From
them, I learned to isolate the mitotic apparatus from sea urchin eggs (pre-
recombinant DNA), film cell dynamics (pre-video) with the DIC microscope (single
molecule microscopy), and investigate the ultrastructure of the dividing sea urchin
egg (pre-cryoTEM). I was exposed to a variety of techniques and learned methods in
cell biology from the best. But little did I know that these summers would lead to a
career-long partnership and friendship with Les in bringing timely methods to a gen-
eration of cell biologists. Now, it is time for Phong Tran and the next generation to
continue this tradition.
MCB has been a team effort. Phyllis Moses brought me to MCB when it was pub-
lished by Academic Press and sponsored by the ASCB. Afterwards, we have worked
with many editors at AP and Elsevier but we shaped the current vision of MCB with
the support of Graham Lees and Jasna Markovac. We were fortunate to expand the
impact of MCB through the resources of Elsevier and our current editor, Zoe Kruze,
and publisher, Lisa Tickner. It’s been a pleasure to have worked with all of them.
Paul Matsudaira
xxiiBiography

CHAPTER
“Stamp-off” to Micropattern
Sparse, Multicomponent
Features
1
Ravi A. Desai*
,{,{,}
, Natalia M. Rodriguez*
,},||
, and Christopher S. Chen*
,},||
*
Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania USA
{
Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany
{
Medical Research Council, National Institute of Medical Research, London, United Kingdom
}
University College London, London, United Kingdom
}
Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA
||
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts,
USA
CHAPTER OUTLINE
Introduction................................................................................................................ 4
1.1 Method................................................................................................................ 5
1.2 Discussion......................................................................................................... 11
Acknowledgments ..................................................................................................... 13
References ............................................................................................................... 13
Abstract
Spatially patterned subtractive de-inking, a process we term “stamp-off,” provides a
simple method to generate sparse, multicomponent protein micropatterns. It has been
applied to control cell adhesion, study adhesion biology, as well as to micropattern
fragile surfaces. This technique can also readily be applied to study nanoscale inter-
actions between cell membrane receptors and surface-immobilized ligands. It is
based on conventional microcontact printing and as such requires the same reagents,
including photolithographically defined masters, a spin-coater, poly(dimethyl
siloxane) (PDMS), and conventional cell culture reagents such as glass coverslips
and adhesive proteins. Stamp-off is conceptually simplified into three steps:
(1) generation of an appropriate cell culture substrate, PDMS-coated glass, (2)
micropatterning with stamp-off, and (3) cell deposition. After elaborating each of
these three methods, we discuss limitations of the technique and its applications.
Methods in Cell Biology, Volume 119 ISSN 0091-679X
Copyright©2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-416742-1.00001-9
3

INTRODUCTION
Cell migration, proliferation, and differentiation are central to a variety of normal
and pathophysiologic processes including embryonic development, tissue homeosta-
sis, wound healing, and cancer progression. In each setting, it appears that there is an
intimate and functional relationship between structure—of the cell and its interaction
with the surrounding microenvironment—and function. Investigating how structure
regulates function on the cellular scale (0.1–10mm) requires a technique to engi-
neer structure while monitoring function at subcellular to cellular length scales
(0.1–10mm). In the past decade, rapid advances in the ability to reliably and effi-
ciently engineer surfaces with geometrically patterned regions presenting adhesive
extracellular matrix surrounded by non-adhesive have led to major insights into how
the structure of the cell, surrounding extracellular matrix, and cell–cell interactions
drive cell functions such as cell life versus death, differentiation, intercellular com-
munication, and migration (Bhatia, Balis, Yarmush, & Toner, 1998; Chen, Mrksich,
Huang, Whitesides, & Ingber, 1997; Connelly et al., 2010; Desai, Gao, Raghavan,
Liu, & Chen, 2009; Dupont et al., 2011; Gilbert et al., 2010; Jiang, Bruzewicz,
Wong, Piel, & Whitesides, 2005; McBeath, Pirone, Nelson, Bhadriraju, & Chen,
2004; Nelson et al., 2005; Nelson, Vanduijn, Inman, Fletcher, & Bissell, 2006;
Thery et al., 2006).
Methods to pattern adhesive surfaces have proliferated rapidly in the past decade,
and many outstanding reviews cover approaches ranging from photopatterning to
microfluidics (El-Ali, Sorger, & Jensen, 2006; Folch & Toner, 2000; Whitesides,
2006). One of the most widely applied micropatterning techniques is microcontact
printing, originally developed by George Whitesides and colleagues over two de-
cades ago (Xia & Whitesides, 1998). In this technique, an elastomeric stamp with
bas-relief features is used to transfer an “inked” material onto a substrate. The elas-
tomer is usually poly(dimethyl siloxane) (PDMS), which has several advantages: (1)
it can readily be made to generate micron-scale features on a substrate of large area (a
few cm
2
)(Kane, Takayama, Ostuni, Ingber, & Whitesides, 1999), (2) PDMS has low
surface energy, enabling it to be easily separated from the template during fabrica-
tion, so binds reversibly to the substance transferred during printing, and therefore
permits easy removal of the stamp from the substrate after printing (Love,
Estroff, Kriebel, Nuzzo, & Whitesides, 2005), and (3) it is relatively inert so does
not react with many chemicals (Xia & Whitesides, 1998). Although microcontact
printing was originally developed to pattern gold (Kumar & Whitesides, 1993), it
was extended to directly micropattern proteins on biocompatible surfaces less than
a decade after George Whitesides pioneered the technique (Bernard et al., 1998;
James et al., 1998) and simplified to enable widespread adoption several years later
(Tan, Liu, Nelson, Raghavan, & Chen, 2004). Thus, microcontact printing rapidly
emerged as a technique of choice to pattern materials for biologic applications.
Despite its utility, conventional microcontact printing suffers from two major
limitations. First, elastomeric stamps bearing small, sparse features are prone to
deformation and collapse during printing, leading to undesired contact of the
4 CHAPTER 1 Stamp-off Micropatterning

inter-feature regions of the stamp with the underlying surface (Ruiz & Chen, 2007;
Xia & Whitesides, 1998). Stamp collapse depends on the pressure applied during
stamping (Hui, Jaogta, Lin, & Kramer, 2002). Investigators have addressed this issue
by using PDMS stamps backed with glass (James et al., 1998), PDMS stamps coated
with a rigid material (Odom, Love, Wolfe, Paul, & Whitesides, 2002), and stamps
made from a material more rigid than conventional PDMS (Schmid & Michel,
2000). However, these strategies still suffer from an upper limit on the spacing be-
tween features, and as such they still place limits on feature design. In general via
conventional microcontact printing methods, the non-adhesive space between pat-
terned features cannot substantially exceed the size of the adhesive features. Thus,
whether and how spacing between cells or the spacing of adhesions within cells af-
fects function are challenging to address via conventional microcontact printing. Sec-
ond, conventional microcontact printing was designed to pattern a single adhesive
ligand, surrounded by a non-adhesive region; the substrates thus present “digital” ad-
hesive cues to cells, and cannot pattern multiple ligands. Multiple ligands can be pat-
terned simply by printing multiple times, but manual spatial registration between
successive printing steps is not trivial (Rogers, Paul, & Whitesides, 1998). Thus,
how cells integrate cues from multiple ligands remains obscure. Although powerful,
conventional microcontact printing suffers from these significant drawbacks.
Here, we describe a method that is a simple extension of conventional microcon-
tact printing to encode a surface with sparse, distinct patterns of multiple proteins.
We describe our technique, called “stamp-off,” in detail and discuss applications
as well as limitations.
1.1METHOD
Stamp-off can be divided into three distinct sections: (1) preparing PDMS-coated
glass, (2) patterning via stamp-off, and (3) seeding cells on the micropatterned sur-
face. Below, we describe these three steps in detail.
I.Preparing PDMS-coated glass.
a.Materials
1.PDMS (Dow Corning, Sylgard 184. Contact Dow Corning for a local
distributor and part number).
2.Glass coverslips. Choose a size and thickness that is compatible with your
application. We typically use 22 mm22 mm square coverslips, number
1.5 thickness (Fisher No. 12-541B. These coverslips fit nicely in a
6-well plate).
3.Transfer pipette (Fisher No. 13-711-7M. Any pipette that will dispense
viscous materials such as uncured PDMS will do. Precise metering of
volume isnotnecessary).
4.Curved tweezers (EMS 0109-7-PO. Any tweezers that will handle coverslips
will do; we find this particular one to be ergonomic and convenient).
51.1Method

5.100% ethanol (Decon Labs, Inc. No. 2716. Need not be pure grade).
6.milliQ water.
7.Compressed nitrogen (e.g., Airgas No. UN1066) with a regulator (VWR
No. 55850-474) and hose and spray nozzle (hose and spray nozzle:
Teqcom No. TA-NS-2000; connector to regulator: Ryan Herco Fluid Flow
Solutions No. 0161.202 5865445).
8.Parafilm (Cole-Parmer No. PM996).
b.Equipment
1.Spin-coater with a chuck to accommodate coverslips (Laurell Technology
Corporation, Model WS-400B-6NPP/LITE; the spin-coater should be
placed in a fume hood or clean room to avoid dust particles during use).
2.Heating oven (optional, only needed to expedite PDMS curing. For
example, Fisher Scientific Isotemp Oven).
c.Method
1.Mix and degas the PDMS as per the manufacturer’s recommendations. We
find that a 10:1 ratio of base to cross linker (by weight) works well for
microcontact printing.
2.Remove a coverslip straight from the container, roughly center on the spin-
coater and apply vacuum. Note that coverslips can be used straight out of
the box without cleaning, but any dust or lint should be blown off with a
stream of nitrogen. In the event coverslips need cleaning, you can sonicate
them in 100% ethanol for 5 min, or shake in 1 N HCl for 15 min at room
temperature. Each method of cleaning requires thorough rinsing with
milliQ water after removing from ethanol or HCl.
3.Use the transfer pipette to dispense roughly 100ml of PDMS onto the
middle of the coverslip. You can eyeball the volume since 100mlis
approximately pea-sized. It is better to drop too much rather than too little
volume, since excess volume will simply spin off but too little volume will
not coat the entire coverslip.
4.Spin the coverslip at 6000 rpm for 60 s. This speed and time will give you a
roughly 15mm PDMS coating.
5.Bake overnight at 60

C. Alternatively, let sit at room temperature for 48 h
to cure.
6.Sterilize
1.Dip the coverslips in 100% ethanol for 5–10 s, careful to coat all
regions of the coverslip with ethanol.
2.Dip the coverslip in a dish filled with milliQ water, and hold for 5–10 s.
Repeat three times.
3.Thoroughly dry coverslip with a stream of N
2.
4.Place coverslip in a 6-well (or appropriate-size) plate.
7.Seal plate with parafilm and store at room temperature until use. PDMS-
coated coverslips can be stored indefinitely at room temperature.
II.Stamp-off. This step assumes that one has already made appropriate stamp-off
templates. SeeFig. 1.1for an example of stamp-off templates. Both the stamps
6 CHAPTER 1 Stamp-off Micropatterning

and stamp-off templates should be made fresh each time this protocol is
performed.
a.Materials
1.Stamp (flat). This is most easily made by casting PDMS off a flat surface
such as a polystyrene dish.
2.Stamp-off template (one for each pattern). This is made by casting PDMS
off a photoresist pattern (a number of very good reviews cover generation
of photoresist patterns, such asWeibel, DiLuzio, and Whitesides (2007)).
FIGURE 1.1
Stamp-off to generate sparse features. (i) First ink the stamp with protein. (ii) Then stamp-off
onto a UV ozone-treated template. (iii) Finally, transfer the protein on the stamp to the cell
culture substrate. The cell culture substrate (also PDMS) should be UV ozone-treated for
7 min. Fluorescent light (FL) micrograph shows an example of corresponding features.
Protein was bovine serum albumin tagged with AlexaFluor488. Scale bar, 20mm. (iii*, iv*)
Sequential re-inking and stamp-off can be performed to generate multicomponent patterns.
SeeFig. 1.2.
71.1Method

3.100% ethanol (need not be pure; 70% ethanol will do).
4.milliQ water.
5.Curved tweezers (EMS 0109-7-PO. Any tweezers that will handle
coverslips will do; we find this particular one to be ergonomic and
convenient).
6.Protein solutions (we typically use fibronectin (BD No. 356008) at
50mg/ml in milliQ water, although a variety of proteins, such as bovine
serum albumin, antibodies, vitronectin, type-I collagen, work as well
(Desai, Khan, Gopal, & Chen, 2011).
7.0.2% (w/v) Pluronic F127 (Sigma No. P2443) in cell culture grade H
2O.
b.Equipment
1.Compressed nitrogen.
2.Laminar Flow Hood.
3.Ultraviolet ozone cleaner (Jelight No. 342. A discussion of a homemade
deep ultraviolet ozone machine can be found inAzioune, Carpi, Tseng,
The´ry, and Piel (2010)).
c.Method. For a visual explanation of conventional microcontact printing, see
Desai, Yang, Sniadecki, Legant, and Chen (2007). Perform this method in a
sterile field.
1.Clean stamps and stamp-off substrates by sonicating for 5 min in 100%
ethanol, dipping in 100% ethanol, dipping in milliQ water, and blowing
dry with a stream of nitrogen, and placing the stamp face-up in a sterile,
dry polystyrene dish.
Note: Avoid touching the “face” of the substrates with tweezers,
gloves, etc.
2.“Ink” the stamp with the desired protein by covering the stamp face with
the protein solution. As long as the stamp face is covered, the volume of
solution does not matter.
Note: The PDMS surface is intrinsically hydrophobic, and this makes
loading the stamp with aqueous solutions difficult. We perform two steps to
address this: (1) pipette a series of approximately 50ml droplets onto the four
corners of the stamp face and let the drops sit undisturbed for 5–10 min (for
illustrative purposes, only one drop is shown inFig. 1.1i, left panel). During
this time, protein should adsorb to the stamp face from the droplets, rendering
the formerly hydrophobic surface hydrophilic at the drop–stamp interface. (2)
Using a fresh pipette tip, “connect the dots” by holding the tip at an angle such
that part of the tip touches the droplet and the other part of the tip touches the
stamp edge. Move the pipette tip along the perimeter of the stamp, careful to
maintain contact between the pipette tip, the droplet, and the stamp edge (see
Fig. 1.1i, middle panel).
3.Incubate the stamps with protein for 1 h at room temperature. Ensure that
the solution does not evaporate.
Note: Depending on kinetics of adsorption, the protein solution may
bead up during this time. If this happens, simply run a pipette tip along the
8 CHAPTER 1 Stamp-off Micropatterning

perimeter of the stamp while maintaining contact with the drop, as in step
(2) above to ensure complete coverage of the stamp (seeFig. 1.1i,
right panel).
4.Activate the stamp-off substrates by placing them in the ultraviolet
ozone cleaner for 7 min at about 5 cm from the ultraviolet light source.
This renders the PDMS hydrophilic for protein transfer. We have
experienced that too little exposure time results in incomplete protein
transfer from the stamp to the stamp-off substrate (see Figure 1 ofDesai
et al. (2011)). An exposure time of 7 min should compensate for
fluctuations in bulb intensity, height, etc.
Note: Be sure to remove the lid of the vessel in which the stamp-off
substrates are placed prior to cleaning with the ultraviolet ozone cleaner, since
most conventional materials such as polystyrene and soda lime glass look
transparent in the visible spectrum but in fact are opaque at deep ultraviolet
wavelengths.
Note: Be sure to remove the lid of the vessel in which the stamp-off
substrates are placed prior to cleaning with the ultraviolet ozone cleaner, since
most conventional materials such as polystyrene and soda lime glass look
transparent in the visible spectrum but in fact are opaque at deep ultraviolet
wavelengths.
5.Rinse the stamps by: (1) pouring milliQ water into the dish containing
the stamps, above the level of the stamps, (2) pick up the stamp and dip it
for 5–10 s in a dish with fresh milliQ water, and (3) dry the stamp
thoroughly with a stream of nitrogen.
Note: It is best to use the stamps immediately after drying, but we have
found that they can be held dry for up to an hour without encountering
problems.
6.Invert the activated stamp-off substrate onto the stamp (Fig. 1.1ii). One
second of contact is sufficient for complete protein transfer.
Note: The stamp-off substrate must be used within 30 min of ozone
treatment. If more than 30 min elapses, the substrate may be re-activated in
the ultraviolet ozone cleaner.
7.Use tweezers to carefully lift the stamp-off substrate from the stamp. Lift
in one smooth stroke to ensure clean pattern edges.
8.Repeat steps 2 (inking) through 7 (stamping) for each additional protein
pattern (Fig. 1.1iii*, iv*). Rotate and/or translate the stamp relative to the
stamp-off substrate as appropriate (seeFig. 1.2iv). Use a new, clean
freshly-activated stamp-off substrate for each stamp-off step.
9.When the stamp bears the desired final pattern, activate the PDMS-
coated coverslips (generated above) in the ultraviolet ozone cleaner for
7 min at about 5 cm from the ultraviolet light source.
10.Invert the stamp onto the activated PDMS-coated coverslip. Press firmly
to ensure conformal contact. Leave in contact for at least 1 s for complete
protein transfer (Fig. 1.1iii).
91.1Method

Note: The stamp is topographically flat so there is no risk of stamp
collapse. Press firmly as needed to ensure conformal contact.
11.Use tweezers to carefully lift the stamp-off substrate from the stamp. Lift
in one smooth stroke to ensure clean pattern edges.
12.Incubate the coverslips in 0.2% Pluronic F127 for at least 1 h at room
temperature.
13.Rinse the substrates three times with milliQ water.
FIGURE 1.2
Stamp-off to generate adjacent, multicomponent features. (i) First ink the stamp with a
protein (green). (ii) Then stamp-off onto a UV ozone-treated template. (iii) Re-ink the stamp
with a second protein (red). The protein should be chosen such that it will adsorb to the bare
PDMS but not to the first protein, seeSection 1.2. (iv) Then stamp-off to remove both the first
and second protein. The stamp-off template should be UV ozone-treated for 7 min. (v) Re-ink
the stamp with a third protein (blue). (vi) Stamp-off to remove the first, second, and third
protein. (vii) Finally, transfer the protein on the stamp to the cell culture substrate. The cell
culture substrate (also PDMS) should be UV ozone-treated for 7 min. Fluorescent light (FL)
micrograph shows an example of adjacent, multicomponent features. Green, red, and blue
proteins were bovine serum albumin tagged with AlexaFluor-488, -594, and -647,
respectively. Scale bar, 20mm.
10 CHAPTER 1 Stamp-off Micropatterning

III.Cell deposition.
a.Materials
1.Phosphate-buffered saline (PBS)
2.Appropriate cell culture medium (for example, DMEMþ10% fetal
bovine serum)
3.Standard cell culture materials (pipettes, centrifuge tubes, vacuum
source, laminar flow hood, cell culture incubator, etc.)
b.Method
1.Detach and resuspend cells as per normal. Adjust cell density to 110
6
cells/ml.
2.Rinse the micropatterned substrate with PBS, and replace the PBS with
cell culture medium.
3.Add cell suspension to the micropatterned substrate at a density of 10,000
cells/cm
2
. Shake plate in perpendicular directions to distribute cells in the
medium.
Note: Shaking the plate by swirling it willnotdistribute the cells, and will
instead force the cells to cluster in the center of the dish.
4.Incubate the micropatterned substrate in an environment appropriate to
the culture conditions (e.g., a humidified incubator set to 37

C and 5%
CO
2for many mammalian cell lines) for 20–60 min until cells have
attached to the micropatterns and begun to spread (for instance, Normal
Rat Kidney-52E’s take about 20–30 min, whereas Human Umbilical
Vein Endothelial Cells take 40–60 min to begin spreading).
5.Remove non-attached cells by very gently aspirating the medium. We
recommend aspirating using a handheld pipet, as vacuum suction can
easily disrupt cells from the micropattern.
Note: Be careful not to dewet the PDMS, as this will cause the Pluronic
F127 to delaminate and/or the cells to dehydrate and therefore lead to pattern
fouling and/or cell death. We find that simultaneously adding media with one
hand while aspirating with the other hand works best to avoid dewetting of
the PDMS.
6.Return the micropatterned substrate to the incubator and proceed with the
experiment (e.g., imaging, lysing and harvesting protein, mRNA, etc.).
1.2DISCUSSION
Stamp-off offers a number of advantages over conventional microcontact printing.
The demonstrated advantages of employing stamp-off to biologic studies are several-
fold. First, the use of multicolor surfaces has been used to interrogate the spatial and
functional relationships of integrin receptors on the cell surface (Desai et al., 2011).
Second, stamp-off has been used to generate sparse features without the risk of stamp
collapse and pattern fouling that stamp collapse entails, since the stamp used in
stamp-off is flat, in contrast to stamps used in conventional microcontact printing
111.2Discussion

(Desai et al., 2011). This permits investigation of whether and how spacing between
cells or the spacing of adhesions within cells affects function. Third, stamp-off has
been used to pattern fragile surfaces such as microfabricated post-array-detectors
(mPADs), owing to the use of a topographically flat stamp (Han, Bielawski, Ting,
Rodriguez, & Sniadecki, 2012; Sun, Weng, & Fu, 2012)(Fig. 1.3A). See
Chapter 5, Vol. 121 for a detailed description of micropatterning mPADs with
stamp-off. Fourth, when different types of cells bind to different surface coatings,
stamp-off also has a potential application in patterned co-culture. For instance, he-
patocytes and fibroblasts bind to different surfaces (type-I collagen and bare glass,
respectively), and this has been exploited by the Bhatia group to generate patterned
co-cultures of these cells. Micropatterned co-cultures were also engineered by the
Chen group to control the juxtaposition of epithelial and mesenchymal cells
(Tien, Nelson, & Chen, 2002). We have used stamp-off to pattern different cell types
(Fig. 1.3B). Finally, cells interact with surfaces via molecular interactions in the
nanoscale regime (Cavalcanti-Adam et al., 2007; Coyer et al., 2012; Paszek et al.,
2012; Schvartzman et al., 2011). Stamp-off, but not conventional microcontact print-
ing, can be used to pattern at the nanoscale to study such interactions (Coyer,
Garcia, & Delamarche, 2007; Xia, Rogers, Paul, & Whitesides, 1999). Despite these
advantages, stamp-off does have several limitations that should be considered when
designing an experiment using stamp-off micropatterning.
First, attention must be paid to ensure accurate spatial alignment.Figure 1.2
illustrates that micropatterns with spatial alignment on the micrometer scale (the
smallest features are 3mm3mminFig. 1.2, and are positioned adjacently without
overlap) can be easily generated, even though actual stamp placement is done by eye
FIGURE 1.3
Applications of stamp-off. (A) A human pulmonary artery endothelial cell seeded on a
micropatterned array of microposts. Blue: DNA, green: actin, red:microposts. Length scale
bar, 20mm. Adapted with permission fromHan et al. (2012)and used with permission from
Cell Press. (B) A three-color pattern, where black represents non-adhesive surface, red
represents an adhesion protein, and green represents a different adhesion protein.Inset:
cells, here expressing cell tracker red or green, segregate to different adhesion proteins. All
scale bars, 20mm.
12 CHAPTER 1 Stamp-off Micropatterning

and only alignment on the millimeter–centimeter scale is required (all alignments in
Fig. 1.2were done by eye). In contrast, to generate the pattern shown inFig. 1.2with
conventional microcontact printing the resolution of stamp placement must be equal
to that required for edge-to-edge spacing. That is, to achieve the same feature sizes
and juxtapositions, one merely needs alignment by eye with stamp-off
(1000–10,000mm), but specialized equipment for alignment of the features them-
selves (typically 10mm or less for micropatterns relevant to cell biology) with con-
ventional microcontact printing.
Second, the inking steps should not use proteins that bind one another. For in-
stance, if protein A binds protein B, then inking the stamp with protein B after
A is already patterned on the surface would allow B to adsorb not only on the
stamped-off (protein free) regions, but also directly to protein A. We have success-
fully micropatterned multicomponent surfaces presenting distinct micropatterns of
fibronectin and protein G (unpublished), and collagen type-I with vitronectin
(Desai et al., 2011) with stamp-off.
Lastly, a limitation to any microcontact printing-based technique such as stamp-
off is that cells can potentially remodel the protein on the surface. This involves cel-
lular digestion of surface-immobilized proteins and discretion and deposition of new
proteins, and the timescale is a function of the proteins involved and cellular enzy-
matic activity, the latter of which depends on the soluble environment (Nelson,
Raghavan, Tan, & Chen, 2003). Thus the timescale of remodeling is largely cell-type
dependent. In contrast, degradation of the non-adhesive region used here (F127
Pluronics) is cell-type independent, has a half-life on the order of weeks (Nelson
et al., 2003).
The power of micropatterning to illuminate fundamental cellular mechanisms is
clear. Given the accessibility and advantages of stamp-off, we hope that the tech-
nique presented herein becomes a valuable tool for interrogating cell functions.
Acknowledgments
This work was supported by grants from the NIH (EB00262, EB08396, and GM74048), the
RESBIO Technology Resource for Polymeric Biomaterials, and Center for Engineering Cells
and Regeneration of the Univ. of Pennsylvania. R.A.D. acknowledges financial support from a
Whitaker International Fellowship. N.M.R. acknowledges support from a National Science
Foundation Graduate Research Fellowship. We thank Rachna Narayanan and Sandra Richter
for careful reading of the manuscript.
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16 CHAPTER 1 Stamp-off Micropatterning

CHAPTER
Poly(Vinyl Alcohol)-
Micropatterned Surfaces
for Manipulation of
Mesenchymal Stem Cell
Functions
2
Guoping Chen
Tissue Regeneration Materials Unit, International Center for Materials Nanoarchitectonics,
National Institute for Materials Science, Tsukuba, Ibaraki, Japan
CHAPTER OUTLINE
Introduction.............................................................................................................. 18
2.1 Preparation of PVA-Micropatterned Surfaces ....................................................... 18
2.1.1 Synthesis of Photo-reactive PVA ........................................................ 18
2.1.2 Micropatterning of PVA ..................................................................... 20
2.2 Effect of Cell Density and Cell–Cell Interaction on Adipogenic, Chondrogenic, and
Osteogenic Differentiation of MSCs ..................................................................... 21
2.3 Effect of Cell Spreading on Adipogenic and Osteogenic Differentiation of MSCs..... 25
2.4 Effect of Cell Geometry on Adipogenic Differentiation of MSCs.............................. 26
2.5 Effect of Cell Protrusion on Adipogenic Differentiation of MSCs ............................ 28
2.6 Effect of Surface Charge on Adipogenic Differentiation of MSCs ........................... 30
Summary .................................................................................................................. 31
Acknowledgments ..................................................................................................... 31
References ............................................................................................................... 31
Abstract
Micropatterning is a useful method to study the effects of biological and physical
cues on cell functions. Various micropatterning methods have been developed for
investigation of cell–cell interaction and cell–material interaction. As one of the
potent methods, poly(vinyl alcohol) (PVA)-based micropatterning has been used
to array cells in a pre-designed manner for a long-term cell culture. Cell population
and single cell arrays can be formed in the micropatterned surfaces. The micropat-
terned surfaces have used to generate a gradient cell density, different degree of cell
Methods in Cell Biology, Volume 119 ISSN 0091-679X
Copyright©2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-416742-1.00002-0
17

spreading, protrusion and cell–cell interaction and different geometry to investigate
their effects on the differentiation of bone marrow-derived mesenchymal stem cells.
This chapter highlights the latest development of PVA-based micropatterning and its
application for manipulation of stem cell functions.
INTRODUCTION
Manipulation of cell functions is important for tissue engineering (Griffith & Naughton,
2002). Various types of stem cells such as embryonic stem cells induced pluripotent
stem cells and mesenchymal stem cells (MSCs) have been established and isolated
for tissue engineering. Differentiation of stem cells into cells of a specific lineage is crit-
ical for regeneration of a tissue or organ.In vivocells are surrounded by their specific
microenvironments (Discher, Mooney, & Zandstra, 2009). The microenvironments
include biological and physiochemical cues to dictate cell migration, adhesion, prolif-
eration, differentiation, and extracellular matrix secretion (Guilak et al., 2009; Pittenger
et al., 1999). Physiochemical cues including size, shape, electrostatic properties, stiff-
ness, roughness and topography havebeenwidelystudied todisclosetheir effects oncell
behaviors, especially on differentiation of stem cells (Connelly et al., 2010; Dalby et al.,
2007; Engler, Sen, Sweeney, & Discher, 2006; Guo, Kawazoe, Fan, et al., 2008;
Higuchi, Ling, Chang, Hsu, & Umezawa, 2013; McBeath, Pirone, Nelson,
Bhadriraju, & Chen, 2004; Peerani et al., 2007). Micropatterning technology is partic-
ularly usefulfor investigatingcellulareffectsofphysiochemicalcuesbecause individual
parameter can be isolated and focused by micropatterning. Micropatterned surfaces and
substrates have been used to investigate cell behaviors such as migration, proliferation,
polarization, and differentiation (Gao, McBeath, & Chen, 2010; Jiang, Bruzewicz,
Wong, Piel, & Whitesides, 2005; Thakar et al., 2009; The´ry et al., 2006). For fabrication
of micropatterned surfaces that are compatible with long-term cell culture to study cell
differentiation, poly(vinyl alcohol) (PVA)-based micropatterning has been adopted
(Kawazoe et al., 2009). The PVA-micropatterned polystyrene surfaces have been used
forstudyingthe effects ofcellspreadingarea, celldensity, cell geometry, surface charge,
cell–cell interaction and cell protrusion on the adipogenic, chondrogenic and osteogenic
differentiation of bone marrow-derived MSCs (Lu et al., 2009; Song, Lu, Kawazoe, &
Chen, 2011a, 2011b, 2011c; Song, Wang, Lu, Kawazoe, & Chen, 2012; Wang, Song,
Kawazoe, & Chen, 2013a, 2013b). This chapter summarizes applications of PVA-based
micropatterns for manipulation of stem cell differentiation.
2.1PREPARATION OF PVA-MICROPATTERNED SURFACES
2.1.1Synthesis of photo-reactive PVA
Photo-reactive PVA is synthesized by coupling some of the hydroxyl groups in PVA
with 4-azidobenzoic acid (Fig. 2.1A). The protocol is as follows (Song et al., 2011b):
18 CHAPTER 2 Poly(Vinyl Alcohol)-Micropatterned Surfaces

FIGURE 2.1
Preparation scheme of photo-reactive PVA (A), micropatterning process using photo-reactive
PVA (B) and proposed photo-reaction mechanism during UV irradiation (C).
192.1Preparation of PVA-Micropatterned Surfaces

1.Dimethyl sulfoxide (DMSO) solution (2 mL) containing
dicyclohexylcarbodiimide (234 mg, 1.13 mol, Watanabe Chemical Industries,
Ltd.) is added dropwise to 5 mL DMSO solution containing 4-azidobenzoic acid
(185 mg, 1.13 mmol, Tokyo Chemical Industry Co., Ltd.) under stirring at room
temperature in the dark.
2.Two milliliter of DMSO solution dissolving 16.8 mg 4-(1-pyrrolidinyl) pyridine
(0.113 mmol, Wako Pure Chemical Industries, Ltd.) is added dropwise to the
reaction mixture under stirring.
3.After 10 min, 8 mL of DMSO solution containing 100 mg of PVA (MW 44,000,
2.26 mmol in monomer units, Wako Pure Chemical Industries. Ltd.) is added
dropwise to the above reaction mixture under stirring in the dark.
4.After 24 h, dicyclohexylurea that formed during the reaction is filtered off. The
filtrate is collected and purified by dialysis against Milli-Q water to obtain the
photo-reactive azidophenyl-derivatized PVA (AzPhPVA).
5.The purified AzPhPVA aqueous solution is freeze-dried to obtain the
AzPhPVA powder.
6.The azidophenyl groups introduced in AzPhPVA are characterized by
1
HNMR.
The percentage of azidophenyl groups in AzPhPVA is determined by
1
HNMR
from the peak intensities of the azidophenyl protons at around 7 ppm and those of
the methylene and methylidyne protons of the polymer main chain at 1.5 and
3.9 ppm, respectively.
2.1.2Micropatterning of PVA
Usually cell-culture polystyrene plate is used for the micropatterning of PVA. The
micropatterning protocol is as follows (Song et al., 2011b):
1.Polystyrene plates (3 cm2.5 cm) are cut from a tissue culture polystyrene flask.
2.AzPhPVA solution is prepared by dissolving the AzPhPVA in pure water at a
concentration of 0.3 mg/mL.
3.0.2 mL of the AzPhPVA aqueous solution is coated on the surface of polystyrene
plates and air-dried at room temperature in the dark.
4.The coated thin layer of photo-reactive PVA is covered with a photomask and
irradiated with ultraviolet light (Funa
®
-UV-Linker FS-1500) at an energy of
0.3 J/cm
2
from a distance of 15 cm (Fig. 2.1B). UV light can pass through the
transparent domains on the photomask and the azidophenyl groups in the photo-
reactive PVA under the transparent domains are activated to generate azido
radicals. The azido radicals react with surrounding molecules to make PVA
molecules under the transparent domains to be inter- and intramolecularly cross-
linked and grafted on polystyrene surface (Fig. 2.1C). The black domains on the
photomask are nontransparent for UV light. After UV irradiation, PVA
molecules under the black domains remain unreacted and can be removed by
washing.
20 CHAPTER 2 Poly(Vinyl Alcohol)-Micropatterned Surfaces

5.After UV irradiation, the plates are immersed in Milli-Q water and ultrasonicated
for 10 min to completely remove any unreacted polymer from the nonirradiated
areas. This process is repeated five times.
6.After washing, the PVA-micropatterned surface is obtained.
The micropatterned surface has a micropattern of polystyrene surrounded with PVA.
The polystyrene micropattern should have cell-adhesive property similar to that of
the cell-culture polystyrene plate. The surrounding PVA regions should inhibit pro-
tein adsorption and cell adhesion. Therefore, cells adhere only on polystyrene re-
gions and follow the polystyrene micropattern to form a micropatterned cell
array. The photomask can be designed to any desirable micropatterns to prepare
PVA-micropatterned polystyrene surfaces having various micropattern structures.
2.2EFFECT OF CELL DENSITY AND CELL–CELL INTERACTION
ON ADIPOGENIC, CHONDROGENIC, AND OSTEOGENIC
DIFFERENTIATION OF MSCs
Cell density has been reported to affect cell functions such as proliferation and dif-
ferentiation. McBeath et al. have reported that human MSCs (hMSCs) seeded at low
density (1000 and 3000 cells/cm
2
) on tissue culture polystyrene tend to differentiate
along the osteoblast lineage whereas hMSCs seeded at high density (21,000 and
25,000 cells/cm
2
) tend to differentiate along the adipocyte lineage (McBeath
et al., 2004). Jaiswal et al. have reported that higher initial seeding density of hMSCs
can significantly enhance mineral deposition, which indicates that the cells have dif-
ferentiated to osteoblasts and have matured (Jaiswal, Haynesworth, Caplan, &
Bruder, 1997). The results of research on the effect of cell density on the differen-
tiation of MSCs in literature seem controversial. For these studies, the effects of cell
density on cell functions have been compared by separately culturing cells at differ-
ent cell density. It is difficult to completely exclude the influence of other factors
induced during separate cell culture from the results. To directly compare the effect
of cell density on cell differentiation, simultaneous culture of cells at various cell
densities on a single surface is desirable.
PVA-based micropatterns have been used to prepare a cell array that has a cell
density gradient on a single surface (Lu et al., 2009; Song et al., 2011b). On the
PVA-micropatterned surfaces, effect of cell density on differentiation of MSCs
can be directly compared. Two types of PVA micropatterns are prepared. One is
stripe micropattern and another is square micropattern. In the stripe micropatterns,
PVA stripes and polystyrene stripes are alternately positioned (Fig. 2.2A). Width of
polystyrene stripes is 200mm and width of PVA stripes from right to left is 20, 100,
200, 400, 600, 800, and 1000mm. When cells are cultured on the stripe micropat-
terns, cells can adhere on polystyrene stripes but not on PVA stripes. Cells on
PVA stripes should move to the polystyrene stripes. By assuming the cells move ran-
domly from the nonadhesive PVA stripes to the adhesive polystyrene stripes, the
212.2Effect of Cell Density and Cell–Cell Interaction

FIGURE 2.2
Illustration of PVA-micropatterned polystyrene surface with a stripe micropattern having
different widths (A), a square micropattern having different areas (B) and a dot micropattern
having different numbers of contact lines (0–3). (C) White indicates polystyrene and gray
indicates surrounding PVA.
22 CHAPTER 2 Poly(Vinyl Alcohol)-Micropatterned Surfaces

final cell density on polystyrene stripes can be theoretically calculated form the
width ratio of PVA stripes and polystyrene stripes. The cell density from the left
to right polystyrene stripes should be 1.10, 1.30, 1.50, 1.75, 2.00, 2.50, 3.00, 3.50,
4.00, 4.50, 5.00, 5.50, and 6.00 times of the seeded cell density. MSCs are seeded
on the PVA stripe micropatterns at a cell density of 510
3
cells/cm
2
and cultured
in serum medium for 3 days. Immediately after cell seeding, the cells distribute evenly
on the PVA-micropatterned surfaces. After 1 day of culture, the cells are observed only
on the polystyrene stripes and formed a striped pattern. Cell density is calculated from
counted cell number. On each stripe from left to right, it is 3.91 to 4.8410
3
,
6.7110
3
, 6.9510
3
, 7.5810
3
, 8.8210
3
, 1.1710
4
, 1.2510
4
, 1.4910
4
,
1.9210
4
, 2.1610
4
, 2.4010
4
, 2.6310
4
, 2.79 to 2.9810
4
cells/cm
2
, respec-
tively. A cell density array from 3.9110
3
to 2.9810
4
cells/cm
2
is formed. After
MSCs form a cell density gradient array for the initial 3 days culture in serum me-
dium, the culture medium is changed to an adipogenic differentiation medium for
adipogenic induction culture for 1, 2, and 3 weeks. Cells are stained with Oil Red O,
a specific marker for adipogenic differentiation. Oil Red O staining shows cells are
positively stained in all the polystyrene stripes. Cells at high cell density are more
densely stained by Oil Red O than the cells at low cell density. Oil Red O staining
also increases with culture time. Although the density of the lipid vacuoles and Oil
Red O staining increase with the increase of cell density, the MSCs at all cell densities
differentiate into adipocyte-like cells. Cell density does not affect adipogenic differ-
entiation of MSCs in the cell density range formed on the stripe micropattern.
To increase the range of cell density, a square micropattern is used (Fig. 2.2B).
A photomask having three dark regions: region A, B, and C, is used to prepare the
micropattern. The length of the side of each dark square is 200mm and the center-to-
center distance between neighboring squares is 400, 1000, and 1414mm for region A,
B, and C, respectively. The dark regions and transparent regions of the photomask
correspond to the polystyrene squares and surrounding PVA regions of the micropat-
terned surfaces. The surface area ratio of polystyrene squares to that of the surround-
ing PVA in region A, B, and C is 1:4, 1:25, and 1:50, respectively. The theoretical
cell density should be 4, 25, and 50 times of the seeding cell density. MSCs are
seeded on the square micropattern surfaces at a cell density of 2.510
3
cells/cm
2
.
After 1 day culture, cell density in the nonpattern region and the regions A, B, and
C is 2.60.6, 10.02.5, 50.015.0, and 112.510.010
3
cells/cm
2
, respectively.
The ratio of cell density in the nonpattern region and the regions A, B, and C to the
seeded cell density is 1.00.2, 4.01.0, 20.06.0, and 45.04.0, respectively.
These results indicate that cell density can be adjusted by controlling the ratio of
cell-adhesive and nonadhesive areas and a cell density gradient with a wide range
(2.6 to 112.510
3
cells/cm
2
) is formed on the square micropattern surface. To in-
vestigate the effect of cell density gradient on the osteogenic differentiation of
MSCs, alkaline phosphatase (ALP) staining is performed after MSCs are cultured
on the PVA-micropatterned surface in osteogenic differentiation medium for 3, 5,
7, and 14 days. The degree of osteogenic differentiation depends on cell density
and culture time as evidenced by ALP staining. At high cell density in region
232.2Effect of Cell Density and Cell–Cell Interaction

C, MSCs are positively stained after 3 days of culture. At medium cell density in
region B, MSCs are only slightly stained after 3 days of culture but positively stained
after 5 days of culture. At low cell density in region A and the nonpattern region,
MSCs are not positively stained after 3 days and only slightly stained after 5 days
of culture but positively stained after 7 days of culture. The relationship between ini-
tial cell density gradient and chondrogenic differentiation is investigated by immu-
nocytochemical staining of type II collagen, which is a specific chondrogenic
marker. Chondrogenesis of MSCs at low cell density in region A and the nonpattern
region is not detected even after 4 weeks of chondrogenic induction culture. MSCs at
medium cell density in region B show negative staining after 2 weeks of culture, but
weak staining after 4 weeks of culture. The staining of MSCs at high cell density in
region C is positive and stable after 2 and 4 weeks of culture.
The results indicate that high cell density initiates osteogenic differentiation more
quickly than does low cell density. The possible reason may be that during the initial
3 days, compared with low cell density, high cell density enhances cell–cell contact,
interaction and thus osteogenic differentiation. The cells at low density show high
proliferation capacity other than osteogenic differentiation. The continuous cell pro-
liferation increases cell density and induces osteogenic differentiation later. There-
fore, the degree of osteogenic differentiation of MSCs is determined by cell density
and culture time. As for the chondrogenic differentiation of MSCs, high cell density
is required.
To further demonstrate cell–cell interaction on differentiation of MSCs, a micro-
pattern is designed to quantitatively control the degree of cell–cell interaction (Wang
et al., 2013a). A photomask having four regions of isolated dots, barbell dots, linear
dots, and honeycomb dots is used to prepare the PVA-micropatterned polystyrene
surface. The diameter of the dots, distance between the dots, and width of the con-
necting lines were 30, 30, and 2mm, respectively (Fig. 2.2C). hMSCs are cultured on
the micropatterned surfaces in control medium. After culture for 6 h, cells adhere
only to the polystyrene dots and not to the nonfouling PVA regions. The dot diameter
is at an appropriate size for single cell adhesion. Single MSCs adhere on the micro-
patterned dots and show different morphologies. The single cells on the isolated dots
remain round and do not spread during culture. The single cells on the barbell, linear,
and honeycomb dots spread slightly. They spread along the connecting lines and be-
come interconnected (direct cell–cell interaction) during the culture period. The sin-
gle cells spread only along the connecting lines, not across the lines. Therefore,
single cells on the isolated dot micropattern have zero cell–cell interaction partners.
The single cells on the barbell, linear and honeycomb dot micropatterns that are
interconnected each other by all the interconnecting lines have one, two, and three
cell–cell interaction partners, respectively.
After culture in osteogenic induction medium for 2 weeks, the cells are stained by
ALP to examine the effect of cell–cell interaction on osteogenic differentiation. The
results show that few cells on the isolated and barbell dots patterns are positively
stained. In contrast, many cells on the linear and honeycomb dots are positively
stained. These results show a clear relationship between osteogenic differentiation
24 CHAPTER 2 Poly(Vinyl Alcohol)-Micropatterned Surfaces

and number of cell–cell interaction. The degree of osteogenic differentiation of
MSCs is 8.41.2%, 9.91.5%, 32.12.3%, and 47.24.3% for cells cultured
on the isolated, barbell, linear and honeycomb dot micropatterns, respectively. Thus,
MSCs on the linear and honeycomb dot micropatterns have significantly higher
potential for osteogenic differentiation than the cells on the isolated and barbell
dot micropatterns. Direct cell–cell interaction is necessary for osteogenic differen-
tiation of MSCs and high number of cell–cell interaction partners facilitates the os-
teogenic differentiation.
2.3EFFECT OF CELL SPREADING ON ADIPOGENIC
AND OSTEOGENIC DIFFERENTIATION OF MSCs
Cell size or spreading area has been demonstrated to affect cell behaviors and func-
tions. Bhadriraju et al. have reported that the stiffness of hepatocytes is affected by
cell spreading (Bhadriraju & Hansen, 2002). Cell stiffness increases with cell spread-
ing but remains low in cells with round morphology. Szabo et al. have demonstrated
the high adipogenic potential of embryonic stem cells on nonadhesive substrata,
where cell spreading is hindered (Szab, Feng, Dziak, & Opas, 2009). However, dur-
ing routine cell culture, the process of cell spreading is often accompanied by a
change in cell shape, the area of cell spreading is diverse among cells and the het-
erogeneity within a cell population, such as cell–cell interaction, is complex. There-
fore, controlling the spreading area of a large number of individual cells with the
same shape is extremely desirable for investigating the effect of cell spreading on
cell functions.
PVA-based micropatterning has been used to control spreading of single MSCs to
investigate the effect of cell spreading on the adipogenic and osteogenic differenti-
ation of MSCs (Song et al., 2011a). A photomask having three different circular
micropatterns with diameters of 40, 60, and 80mm is used for micropatterning of
PVA on polystyrene surfaces. Three different circular polystyrene micropatterns
are formed and surrounded by PVA domains after micropatterning (Fig. 2.3). MSCs
are cultured on the micropatterned surfaces. After 6 h of culture, MSCs only adhere
on cell-adhesive circular polystyrene micropatterns and MSCs on nonadhesive PVA
regions are removed by a medium change. About 85% of circular micropatterns are
occupied by a single cell. Therefore, the heterogeneity of the cell population in rou-
tine cell culture can be reduced and the behaviors and functions of MSCs can be stud-
ied at a single cell level.
MSCs are cultured on the micropatterns in adipogenic induction medium for 7
days. Lipid vacuoles are observed in some MSCs on the micropatterns. Lipid vac-
uoles are stained with Oil Red O. The probability that MSCs with different degrees
of cell spreading commits to adipocytes is evaluated. Only single cells on each cir-
cular micropattern are counted. The results show that the probability of MSC adipo-
genesis is dependent on the degree of cell spreading. The percentages of MSCs
undergoing adipogenic differentiation are 45.33.4%, 26.33.4%, and 14.74.2%
252.3Effect of Cell Spreading

for 40, 60, and 80mm circles, respectively and 12.42.0% for the bare polystyrene
surface (nonpatterned). Therefore, the probability of adipogenic differentiation of
MSCs decreases as the degree of cell spreading increases.
Osteogenic differentiation of MSCs on the micropatterns is evaluated by cultur-
ing in osteogenic induction medium for 7 and 21 days. Osteogenic differentiation is
evaluated by ALP staining. The percentages of MSCs undergoing osteogenic differ-
entiation are 13.02.2%, 28.33.0%, and 41.21.9% on micropatterns with 40,
60, and 80mm circles, respectively and 54.64.2% on the bare polystyrene surface
(nonpatterned) after 7 days of osteogenic induction culture. The probability of oste-
ogenic differentiation of MSCs increase as the degree of cell spreading is enhanced.
After osteogenic induction culture for 21 days, the percentages of MSCs undergoing
osteogenic differentiation are 17.53.5%, 40.23.8%, and 53.95.4% on the
micropatterns with 40, 60, and 80mm circles, respectively and 86.03.0% on the
bare polystyrene surface (nonpatterned). Although the trend of the probability of
osteogenic differentiation at 21 days is similar to that at 7 days, more cells undergo
osteogenic differentiation after long-term culture. These results indicate that cell
spreading facilitates osteogenic differentiation of MSCs.
2.4EFFECT OF CELL GEOMETRY ON ADIPOGENIC
DIFFERENTIATION OF MSCs
As previously shown, cell size and spreading area can affect adipogenic and osteo-
genic differentiation of MSCs. High degree of cell spreading favors osteogenic dif-
ferentiation while low degree of spreading favors adipogenic differentiation of MSCs.
The results are similar to those reported byMcBeath et al. (2004). In their report, when
MSCs are cultured in single form in a mixture medium of adipogenic and osteogenic
FIGURE 2.3
Illustration of micropatterned polystyrene dots with different diameters of 40, 60, and 80mm.
White indicates polystyrene and gray indicates surrounding PVA.
26 CHAPTER 2 Poly(Vinyl Alcohol)-Micropatterned Surfaces

differentiation, MSCs prefer to differentiate to adipocytes on small square island
(1024mm
2
) but to osteoblasts on large square island (10,000mm
2
). Besides the effect
of cell size, cell shape or geometry has also reported to affect differentiation of MSCs.
Falconnet et al. have reported that intermediate cell size (2500mm
2
) shows little bias
in directing cell differentiation toward either osteogenic or adipogenic fates
(Falconnet, Csucs, Grandin, & Textor, 2006). Interestingly, at such intermediate size,
the geometry of the cell adhesion area can direct the differentiation of MSCs. Rect-
angle with high aspect ratio and star shape lead to preferential osteogenesis. In con-
trast, square with low aspect ratio and flower shape favor adipogenesis. However, it is
not clear whether different geometries with a small surface area have effect on the
adipogenesis of MSCs in adipogenic induction medium alone.
Therefore, a PVA micropattern having different geometry of small size is used
for culture of MSCs to study the effect of cellular geometry on adipogenic differen-
tiation of MSCs. A photomask having different geometric regions: regular triangle,
pentagon, hexagon, square and circle, is used to prepare the micropatterned surface.
After micropatterning, five types of polystyrene geometric micropatterns are formed
and surrounded by PVA domains on a polystyrene cell culture plate (Fig. 2.4). MSCs
adhere on the micropatterns and spread following the underlying geometric micro-
patterns. About 90% of the geometric micropattern domains are occupied by a single
cell. Therefore, stem cell functions related to cellular shape can be investigated at the
single cell level while excluding the effect of cell–cell interactions and cell density.
After culture in adipogenic induction medium for 7 days, the cells on the five types of
micropatterns remain viable and keep the same shapes as the cells cultured for initial
6 h in control medium. Lipid vacuoles are observed in some of the cells.
The cells after culture in adipogenic induction medium for 7 days are stained by
Oil Red O. The influence of different cellular shapes on the probability of MSCs
adipogenesis is compared by calculating the percentage of MSCs that are committed
FIGURE 2.4
Illustration of micropatterns of different shapes having the same areas (1134mm). White
indicates polystyrene and gray indicates surrounding PVA.
272.4Effect of Cell Geometry on Adipogenic Differentiation of MSCs

to an adipocyte lineage. The percentage of adipogenic differentiation of MSCs is
35.71.4%, 33.95.5%, 35.84.7%, 43.02.5%, and 42.96.3% on the triangu-
lar, square, pentagonal, hexagonal, and circular micropatterns, respectively. Although
the MSCs with hexagonal and circular shapes show slightly higher potential for adipo-
genesis, there is no significant difference among different cellular shapes. However, the
percentage of adipogenic differentiation of MSCs on a nonpatterned surface is signif-
icantly lower than that on the micropatterns, which is consistent with the report that a
smaller spreading area favors the adipogenic differentiation of MSCs.
The five types of geometric micropatterns (triangle, square, pentagon, hexagon,
and circle) have varying degrees of roundness, but all are symmetrical and small for
cell spreading. Based on the formation of lipid vacuole, no significant difference in
the adipogenic differentiation of MSCs is detected among these five geometric
micropatterns. However, MSCs show significantly higher potential for adipogenic
differentiation on these micropatterns than on the nonpatterned polystyrene surface.
Low cell density is used in this study to ensure high possibility of single cell to be
located in each micropattern. By comparing cells on the micropatterns and nonpat-
terned surface, cell spreading is suppressed by the micropatterns, which have some
positive effect on the adipogenic differentiation of MSCs. The result that constrain-
ing MSCs in symmetrically small micropatterns favors adipogenic differentiation of
MSCs is similar to the results reported byMcBeath et al. (2004).
It has been reported that cytoskeletal organization in cells is important to the com-
mitment of MSCs (Falconnet et al., 2006). Generally, the assembly of cytoskeleton
correlates with intracellular contractility. Large cell spreading and increased contrac-
tility favor osteogenic differentiation, while small cell spreading and low contractil-
ity favor adipogenic differentiation. MSCs cultured on triangular, square,
pentagonal, hexagonal, and circular micropatterns in control medium show similar
patterns of actin filaments. Although actin filaments are thicker and denser at the
edges than in the interior regions of the micropatterns, asymmetrical concentrations
of actin filaments are not shown in either edge of all the geometries and no predo-
minated alignment of the actin filaments emerges inside the micropattern geometry.
After culture in adipogenic induction medium for 1 week, the actin cytoskeleton un-
dergoes remodeling and the differentiated cells show faint actin filaments. The sim-
ilarity and symmetry of cytoskeletal structures may implicate the similar low level of
intracellular contractility in the cells cultured on the five micropatterns and may par-
tially cause the parallel potential of adipogenic differentiation of MSCs on the micro-
patterns. Another possibility is that the global low contractility in cell is dominated
by same small cell size rather than different cell shapes.
2.5EFFECT OF CELL PROTRUSION ON ADIPOGENIC
DIFFERENTIATION OF MSCs
Except cell spreading area, cell morphology and cell–cell interaction, the effect of
cell protrusion on the differentiation of stem cells has also be investigated by using
PVA-based micropatterns (Wang et al., 2013b). Three photomasks having different
28 CHAPTER 2 Poly(Vinyl Alcohol)-Micropatterned Surfaces

dot size (30, 50, and 70mm) and different number of 2mm-wide and 30mm-long pro-
trusion line (0, 1, 2, 3, 4, and 6 short lines) are used. The micropatterns are used for
culture of MSCs to obtain single cell micropattern with different spreading area and
different degree of protrusion. The cells adhere on the micropatterned dots and
spread along the protrusion lines following the micropatterns. The cell morphology
can be manipulated by the micropatterns.
MSCs on all the micropatterns show positive staining of Oil Red O, which sug-
gests that MSCs on all the micropatterns undergo adipogenic differentiation. The
influence of spreading area and number of protrusion lines on adipogenic differen-
tiation of MSCs is compared by calculating the percentage of positively stained
cells on the different micropatterns. When MSCs are cultured on the micropatterns
having central dot size of 30mm, about 60% of the MSCs undergo adipogenic dif-
ferentiation after culture for 7 days. The cells on the micropatterns having 4 and 6
protrusion lines show significantly lower percentage of adipogenic differentiation
than the cells on the micropatterns having 0, 1, 2, and 3 protrusion lines. The cells
on the micropattern having six protrusion lines show the lowest percentage of adi-
pogenic differentiation. When the adipogenic induction culture time increases to
14 days, the percentage of cells showing adipogenic differentiation increases
slightly. However, MSCs show only about 50% and 35% of adipogenic differenti-
ation after 7 days culture on the micropatterns having central dot size of 50 and
70mm, respectively. The percentage of adipogenic differentiation increases signif-
icantly after 14 days culture. There is no significant difference among the micro-
patterns having different number of protrusion lines, suggesting that the number of
protrusion lines does not affect adipogenic differentiation when the central dot size
was large (50 and 70mm). The results indicate that the size of the micropatterns has
a predominant influence on adipogenic differentiation of MSCs while the number
of protrusion lines has a micropattern size-dependent influence. The number of
protrusion lines can affect adipogenic differentiation when the micropattern size
is small.
The effect of the central dot size and protrusion lines can be explained by the
change of F-actin structure induced by the micropatterns. F-actin structure has
been reported to be related with the differentiation of MSCs (Kilian, Bugarijia,
Lahn, & Mrksich, 2010). Small spreading area has been reported to facilitate adi-
pogenic differentiation because of less stress of F-actin. The MSCs on the small
dot micropatterns (30mm) show less degree of organization and lower stress of
F-actin than the cells on the large dot micropatterns (50 and 70mm). This may
be of the reasons that the central dot micropatterns with 30mm diameter promote
adipogenic differentiation of MSCs more strongly than the dot micropatterns hav-
ing 50 and 70mm diameter. The number of protrusion lines shows significant
effect on adipogenic differentiation when the micropattern is small. This may
be explained by the increase of organization degree and stress of F-actin when
the number of protrusion line increases to 4 and 6. The effect of number of pro-
trusion lines on the adipogenic differentiation of MSCs on the large micropatterns
(50 and 70mm) is not evident, which may be due to the high stress of F-actin no
matter with or without protrusion.
292.5Effect of Cell Protrusion on Adipogenic Differentiation of MSCs

2.6EFFECT OF SURFACE CHARGE ON ADIPOGENIC
DIFFERENTIATION OF MSCs
Surface chemistry is a critical property of biomaterials and scaffolds because it
affects the surface energy, wettability, and charge. It has been reported that methyl
group maintains the phenotype of MSCs (Benoit, Schwartz, Durney, & Anseth,
2008); amino, thiol and phosphate groups promote osteogenesis (Benoit et al.,
2008; Curran, Chen, & Hunt, 2005; Guo, Kawazoe, Fan, et al., 2008; Guo,
Kawazoe, Hoshiba, et al., 2008); carboxyl and hydroxyl groups facilitate chondro-
genesis (Benoit et al., 2008; Guo, Kawazoe, Hoshiba, et al., 2008; Lanniel et al.,
2011). Although these works discern the respective effects of the different chemical
groups on the differentiation of MSCs, the results are derived from responses across
multiple cell populations cultured under conventional conditions. Under these con-
ditions, cells freely spread and divide in all directions, resulting in an inherent var-
iability in cell spreading, shape, and behavior; therefore, results are sometimes
controversial. The high cell-to-cell variation of the overall population highlights
the importance of single cell analyses regarding the effects of surface chemistry.
A method to prepare micropatterned surfaces with different surface chemistry has
been developed by using photo-reactive polymers (Guo, Kawazoe, Fan, et al., 2008;
Guo, Kawazoe, Hoshiba, et al., 2008). Negatively charged poly(acrylic acid) (PAAc)
micropatterns and neutral polystyrene micropatterns are prepared using photolithog-
raphy. The micropatterns are used to study single cell cultures of MSCs and they
allow for the control of shape and spreading of the MSCs. Using this method, the
electrostatic effect on the functions of individual MSCs is investigated.
Photo-reactive PAAc is at first synthesized by coupling the carboxyl groups of
PAAc with 4-azidoaniline. Photo-reactive PAAc is then grafted to the surface of
cell-culture polystyrene plate using UV irradiation without a photomask. To prepare
the PAAc micropatterns, the PAAc-grafted plate is coated with photo-reactive PVA
and dried. The PVA-coated PAAc-grafted polystyrene surface is covered with a pho-
tomask and irradiated with UV light. The photomask has three different circular micro-
patterns with diameters of 40, 60, and 80mm. Three differently sized circular
micropatterns of PAAc are formed and surrounded by PVA, which can effectively pre-
vent cell adhesion. The PVA-micropatterned surface is prepared by directly micropat-
terning photo-reactive PVA onto the polystyrene surface using the same photomask.
After culturing MSCsonthe micropatternsfor6 h inthe control medium, MSCsonly
adhere to the circular PAAc and polystyrene micropatterns; the MSCs on the nonadhe-
sive PVA regions are removed by changing the medium. These results confirm that the
MSCs are confined within the circular micropatterns on the micropatterned surfaces.
The MSCs are cultured on the PVA-PAAc-micropatterned and PVA-
micropatterned PSt surfaces in the adipogenic medium for 7 days. Some MSCs
on the micropatterns differentiate into adipocytes containing lipid vacuoles. The
effects of surface charge and circular diameter on adipogenic differentiation of
MSCs are evaluated. The probability of the adipogenesis of the MSC is dependent
on the surface charge and circular diameter. At the single-cell level, the adipogenesis
30 CHAPTER 2 Poly(Vinyl Alcohol)-Micropatterned Surfaces

of the MSCs is enhanced on the negatively charged PAAc micropatterns when the
circular diameter is small (40 and 60mm micropatterns). By contrast, the adipogen-
esis of the MSCs is similar between the negatively charged PAAc and the neutral PSt
micropatterns when the circular diameter is large (80mm micropatterns). Moreover,
the percentage of differentiated MSCs decreases as the circular diameter increases;
this trend is independent of surface charge.
Atthesingle-celllevel,thesmaller,negativelychargedPAAc micropatternsenhance
adipogenic differentiation. However, the enhancement effect of PAAc is not obvious
in the large micropatterns; the effect of PAAc may be counteracted by the area effect,
as the adipogenic differentiation decreases when cell spreading increases. One possible
explanation for the stimulatory effect of the negatively charged PAAc micropatterns
may be proteins adsorbed from the medium or from the secreted extracellular matrix.
SUMMARY
PVA-based micropatterning has been used for investigation of the effects of various
physiochemical cues impact on adipogenic, chondrogenic, and osteogenic differen-
tiation of hMSCs. Cell array having different cell density on a single surface can be
obtained by tailoring ratio of cell adhesive area and nonadhesive area. Single cell
arrays having controlled cell–cell interaction, different spreading area, geometry,
protrusion and surface chemistry can be realized by using the PVA-micropatterned
surfaces. Comparison of differentiation of MSCs on these single arrays discloses
some detailed information of the effects of these physiochemical cues on the com-
mitment of MSCs. The PVA-micropatterned surfaces are stable during cell culture
and the cell arrays can maintain the micropattern structures during long-term culture.
The micropatterning method will be useful to construct diverse micropatterns on var-
ious substrates with not only physiochemical cues but also biological cues to inves-
tigate the corresponding effect of these factors on stem cells.
Acknowledgments
This work was supported by the World Premier International Research Center Initiative on
Materials Nanoarchitectonics from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
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33References

CHAPTER
Patterning of Polymeric Cell
Culture Substrates
3
Alexander Welle*
,{
, Simone Weigel
{
, and O¨zgu¨l Demir Bulut
{
*
Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany
{
Institute for Biological Interfaces, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany
CHAPTER OUTLINE
Introduction.............................................................................................................. 36
Patterning of Self-Assembled Monolayers................................................................... 37
Polymer Patterning.................................................................................................... 38
3.1 Equipment and Protocols .....................................................................................39
3.1.1 Safety and General Recommendations ...............................................39
3.1.2 The UV Lamp ...................................................................................39
3.1.3 UV Dosimetry ...................................................................................40
3.1.4 Lithography Masks............................................................................40
3.1.5 Substrates .......................................................................................41
3.1.5.1 Protocol for Spin Coating of Si Wafers or Glass with PS Films......41
3.1.6 UV Irradiation ..................................................................................42
3.1.7 Cell Culture and Media Supplements .................................................42
3.1.8 Advanced Technology .......................................................................43
3.1.8.1 Protocol.....................................................................................43
3.1.9 Problems and Workarounds ...............................................................43
3.1.9.1 Proximity Gap............................................................................43
3.1.9.2 Inhomogeneous Seeding............................................................43
3.2 Results and Discussion .......................................................................................44
3.2.1 Surface Characterization ...................................................................44
3.2.2 Cell Adhesion...................................................................................47
3.2.3 Competitive Protein Adsorption..........................................................50
Conclusions.............................................................................................................. 51
Acknowledgments ..................................................................................................... 51
References ............................................................................................................... 52
Methods in Cell Biology, Volume 119 ISSN 0091-679X
Copyright©2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/B978-0-12-416742-1.00003-2
35

Abstract
The purpose of this chapter is to provide a summary of polymer patterning technol-
ogies for biological applications and detailed instructions for resist-free deep ultra-
violet (UV) patterning of poly(styrene). Photochemical modifications of this
polymer yield unstable peroxides together with stable oxidized chemical groups.
The altered physicochemical properties of the polymer surface influence protein
adsorption and cell adhesion. HepG2 (human hepatoma cell line), fibroblasts
(L929, murine fibroblast line), and other cell lines exhibit strong adhesion on areas
of UV-irradiated polymer. Masked irradiations open a simple, fast (cell patterns are
obtained within a few hours), and economical route to obtain chemically patterned
cell culture substrates. The described protocol is advantageous compared to silane-
based patterning techniques on glass or thiol-based patterning on gold because of the
elimination of any chemical treatment and the small size of achieved structures. The
protocol is compatible with common clean room technologies; however, even with-
out access to a clean room, structured substrates can be produced. The described
technique can be a useful tool for a variety of cell cultures used to study biological
processes like intercellular communication and organogenesis and for applications
like biosensing or tissue engineering.
Most of the material of our bodies and brains, after all, is being continuously
replaced, and it is just its pattern that persists. (Penrose, 1994)
INTRODUCTION
Without powerful patterning techniques at the sub-micrometer scale, this text would
have been written with a pen or on a typewriter. However, micro-lithographies com-
bined with silicon chip processing and software developments paved the way for
computing and keep changing sciences and our daily lives at increasing speed. It is
noteworthy that the old fashion pen, typesetting, and stamping became miniaturized
and are currently used in high-tech fields, such as dip pen lithography and micro-
contact printing,v. inf., and are applied also for the patterning of cell culture substrates.
The overall goal of cell patterning is to overcome the limitations of conventional
cell cultures. Unstructuredin vitrocultures are often too far from the complex and
microscaled architectures foundin vivo. Even patterned substrates do not avoid some
problems associated with two-dimensionality and bulk properties of the cell culture
substrate; however, they provide helpful tools for basic research. For a review see
Alves, Pashkuleva, Reis, and Mano (2010).
One example showing the differences between conventional and micro-patterned
cell cultures is provided by Sangeetha Bhatia (1999) at the Laboratory for Multiscale
36 CHAPTER 3 Patterning of Polymeric Cell Culture Substrates

Regenerative Technologies, MIT: It was clearly demonstrated that patterned cell cul-
tures from rat hepatocytes together with 3T3-J2 fibroblasts exhibited increased liver
metabolism (albumin secretion and urea synthesis) (Bhatia, Balis, Yarmush, &
Toner, 1998a). Not only can the stabilization of cellular metabolism and phenotype
be accomplished on microstructured scaffolds but also the differentiation of human
mesenchymal stem cells. It was shown that geometric cues can be an important part
of the cellular microenvironment guiding cell differentiation (Kilian, Bugarija,
Lahn, & Mrksich, 2010).
It should be noted here that living cells will not interact directly with a man-made
surfacein vitrobut rather with the protein film deposited on top of this surface.
Therefore, controlling the physicochemical properties of a “bio interface” allows
the building of protein patterns and, therefore, the guidance of cell adhesion and
further cellular processes like metabolism, differentiation, or apoptosis. On the basis
of this understanding, numerous groups have developed a broad variety of patterning
protocols. They differ strongly with respect to obtained lateral resolutions, involved
substrate chemistries, and patterning methodologies. Hence, the following overview
is not exhaustive.
A patterning protocol presented by Bhatia and colleagues (Bhatia, Balis,
Yarmush, & Toner, 1998a, 1998b) shows design similarities to conventional
microelectronic lithographies: Many processing steps like photoresist deposition,
exposure, development, and stripping also are found in semiconductor production
but adapted to protein and cell patterning. With respect to the substrate materials,
there are two large classes of described patterning protocols.
PATTERNING OF SELF-ASSEMBLED MONOLAYERS
With the increasing popularity of molecular monolayers of thiols on gold or other
coinage metals and silanes on silicon or glass, special patterning technologies for
those thin layers emerged. Thiol self-assembled monolayers (SAMs) have been
patterned with a variety of technologies: micro-contact printing (Mrksich, 2000),
nanografting, i.e., the locally controlled deposition of SAMs on gold (Liu,
Amro, & Liu, 2008), as well as lithographies based on conventional, masked ultra-
violet (UV) (300 nm) exposure (Driscoll, Milkani, Lambert, & McGimpsey, 2010),
scanning near-field photolithography (Leggett, 2006), with very localized irradiation
from a scanning near-field optical microscope coupled to a UV laser, or electron
beam exposure (Zhou, Trionfi, Jones, Hsu, & Walker, 2010). The strengths of some
of the aforementioned technologies are very high lateral resolutions. In principle,
lateral resolutions down to the 1 nm range are possible; however, at that scale,
surface analysis is equally as complex as the patterning itself. Zhou et al. report edge
resolutions of 1.3mm. The review of Leggett lists patterns with structure sizes well
below 100 nm. These high-resolution approaches require sophisticated instrumenta-
tion and are usually beyond the means of many laboratories.
37Patterning of Self-Assembled Monolayers

POLYMER PATTERNING
Starting from conventional tissue culture substrates, that is, poly(styrene) (PS), and
other polymers, patterning can be performed. PS remains the most convenient ma-
terial for cell biology because of its low cost, biocompatibility, and its compatibility
with usual microscopic techniques. It is extensively used in cell culture experimen-
tation in the commercially available form of Petri dishes, multiwell plates, and so on.
Several polymer patterning technologies are available: Plasma processing of
polymers can be applied (Lehmann et al., 2010). For plasma processing, the direct
contact of radicals and excited neutrals from the nonequilibrium plasma phase with
the polymer surface must be allowed. Therefore, conventional glass masks for pho-
tolithography cannot be applied; instead, masks made from PDMS having grooves,
pits, or open channels allowing the access of the plasma are applied (Pelzl, Arcizet,
Piontek, Schlegel, & Heinrich, 2009), or a combination of resist lithography
and oxygen plasma treatment is performed (Dupont-Gillain, Alaerts, Dewez, &
Rouxhet, 2004). It is reported that the surface hydrophilicity of PS introduced by
plasma treatments decays with time. Plasma treatment was shown to introduce chain
scission and, thus, the formation of low molecular weight fragments, which enable the
polymer to recover some hydrophobic character; seeKtari et al. (2010)and references
therein. In contrast, corona treatment, applied in the production of conventional PS
tissue culture dishes, yields very stable modifications of the polymer surface. Another
patterning protocol, (Ktari et al., 2010), is based on a scanning technology: Patterning
PS and a cycloolefin copolymer by scanning electrochemical microscopy.
The review ofGonzalez-Macia, Morrin, Smyth, and Killard (2010)presents a
valuable overview of further technologies (e.g., laser, inkjet, screen, gravure, and
flexographic printing).
A rather new class of patterning technologies is designed for multicolor printing:
Dip pen and polymer pin lithography (Brinkmann et al., 2013) and DNA-based pat-
terning, exploiting the effective and highly selective hybridization of complementary
oligonucleotide strands (Gandor et al., 2013; Reisewitz et al., 2010).
Finally, polymer patterning protocols can be mask-based, resist-free photolithog-
raphies (Montero-Pancera et al., 2010; Oliveira, Song, Alves, & Mano, 2011).Yang
and Yang (2013)characterized the UV lithography of PS and similar technologies
by the following features: “Compared with many other methods, photochemical re-
actions have fast rates, high efficiencies, and environmental friendliness. The pen-
etration depth of UV light is dependent on the wavelength and incident angle.
Top-down techniques such as photochemistry, which facilitate the preparation
of gradient surfaces, have found a wide range of applications in high-tech fields.
Another typical characteristic of photochemistry is the tunable input energy for
CdH activation based on the selection of UV light with different wavelengths, in-
tensities, and irradiation times as well as irradiation distance, a thickness of
UV-absorbing material or solution. Longer wavelengths, lower intensities, shorter
irradiation times, longer irradiation distance, or thicker UV-absorbing layer result
in a lower input energy at the surface andvice versa.”
38 CHAPTER 3 Patterning of Polymeric Cell Culture Substrates

The obvious drawback of any mask-based lithographic process is the limited flex-
ibility of patterns. For new patterns, a new mask must be designed and obtained from
external suppliers. Writing patterns with a scanned laser beam is more flexible but
requires very expensive equipment compared to the mask-based lithography that is
presented in the following section.
3.1EQUIPMENT AND PROTOCOLS
Using the described protocol, we have successfully patterned a variety of different
cell lines: L292 fibroblasts, HepG2 hepatoma cells, rat pheochromocytoma cells
PC-12, H9c2 myoblasts, and others. The presented patterning technology is fast
and straightforward. Some micrographs of cell and protein patterns were obtained
during a course for undergraduate students.Polymer surface patterningbased on
deep UV light requires no photo sensitizers and can be scaled up easily to substrates
of some 10 cm
2
. Apart from the photo masks, no expensive equipment is required.
The conventional lithography masks are produced by e-beam writing that allows for
sub-micrometer patterns on the masks. The lateral resolution of a simple tabletop UV
exposure setup is in the range of a few micrometers due to the inevitable proximity
gap between the photo mask and the substrate. Without special vacuum holders and
other specialized equipment, this small gap, together with the size of the UV lamp,
limits the lateral resolution. However, this might be used to obtain chemical gradi-
ents on the substrate because finely structured masks plus diffuse UV illumination
allow for gradient exposures.
3.1.1Safety and general recommendations
Ultraviolet light skin exposure can result in sunburn and skin cancer. Wear protective
clothing (thin disposable surgery gloves) and UV blocking safety glasses. UV with
l<254 nm produces ozone in air. Because ozone is a harmful gas, the UV exposure
should be carried out in a fume hood. A lowered front window usually also blocks
UV irradiation. UV and ozone degrade organic material; polymers can become yel-
lowish and brittle. Therefore, electronic equipment, cables, and other sensitive
materials should be shielded from prolonged UV irradiation.
Like every small scale patterning technique,deepUVli
thographyis hindered by
dust particles; however, a special clean room might not be necessary if substrates and
lithography masks are handled with care. Clean the lithography masks prior to use
with a solvent such as acetone followed byi-propanol and blow dry with a nitrogen
stream. Do not use compressed air because it might contain oil traces.
3.1.2The UV lamp
Light of a wavelength between 400 nm (violet) and 10 nm (X-rays) and energies
from 3 to 124 eV is termed UV. The ISO-21348 subdivides UV light according to
wavelengths: UVA, 400–315 nm; UVB, 315–280 nm; and UVC, 280–100 nm.
393.1Equipment and Protocols

It is important to use a lamp that emits UVC, also termed “far UV,” “deep UV,” or
“vacuum ultraviolet,” (VUV)! These lamps are usually low-pressure mercury
vapor lamps.
Note: High-pressure mercury lamps installed at most fluorescence microscopes
are not suitable because they do not emit the short wavelengths needed for this
lithography. Please check the manufacturer’s data sheet to ensure that the lamp is
emitting 185 nm. The term “ozone-generating lamp” is often used.
UV-Consulting Peschl, Mainz, Germany, offers suitable UV lamps (NNQ series),
power supplies, and accessories. NNQ lamps, as well as some other mercury lines,
emit UV at 185 and 254 nm. A fully closed housing or at least a reflector for this lamp
is required from local apparatus manufacturing. Bioforce Nanosciences, Inc., Ames,
Iowa, USA, sells a small UV cabinet, UV/Ozone ProCleaner
™
. This small device
comes ready to be used; however, it allows no UV intensity adjustment and provides
very diffuse light, limiting the lateral resolution.
3.1.3UV dosimetry
For the beginning, UV dosimetry seems unnecessary; however, it is indispensable for
the comparison of experiments performed in several runs and, more important, to
allow for comparability with published data of other groups. Often, authors provide
information on the type of lamp, the distance to the substrate, and the electrical power
consumption (input) of the lamp. This power consumption (in the range of some
10 W) must not be confused with the actual UV intensity output at the sample po-
sition (some 100mW/cm
2
) and provides no accurate process description.
Some experimental effects can be corrected with the careful application of UV
dosimetry:
a.Lamps
are heating up during the first 10–20 min after ignition, and, during that
time, the UV output is not stable.
b.Lamps do show aging effects and gradually lose their brightness.
c.Fingerprints and other contaminations on the lithography mask, the quartz tube of
the lamp itself, or the reflector reduce UV light intensity on the sample.
Hamamatsu offers N.I.S.T. traceable UV dosimeters for different wavelengths.
3.1.4Lithography masks
Together with the UV source, the lithography mask is the key element of this pat-
terning process. Chromium/quartz lithography masks that can be obtained from a va-
riety of manufacturers are usually applied in semiconductor industries. After
providing a suitable computer aided design file (commonly “Graphic Data System
II” files, GDS; or “Drawing Interchange File” format, DXF), any pattern can be
obtained with feature sizes smaller than 1mm. Structures are defined by their out-
lines; therefore, information about whether closed lines should become chromium
or transparent areas must be given. Further information is needed in case of hierar-
chical patterns (a ring is defined by two concentric circles) and if mirroring is
40 CHAPTER 3 Patterning of Polymeric Cell Culture Substrates

requested. Masks are offered from 4
00
to 7
00
in size or can be cut to smaller pieces on
request. Make sure that the substrate is deep UV transparent quartz; cheaper boro-
silicate or soda lime glass cannot be used.
Looking at the mask from the structured side that must be placed directly onto the
PS surface, the patterned metal layer appears brownish. Viewed from the other side,
the chromium layer on the quartz slide appears silver. Make sure to place the lithog-
raphy mask with the brown side facing the substrate, otherwise no micro-pattern will
be obtained. Lithography masks are fairly robust but should be handled with twee-
zers or special fine pen-type vacuum grippers (available from suppliers for electron-
ics and automation) and cleaned with acetone andi-propanol if necessary.
3.1.5Substrates
The most suitable substrates for the presented patterning technology are disposable,
sterile bacterial culture Petri dishes. These dishes are cheap, very widely used, avail-
able in different sizes, clean, and dust free. They are manufactured from pure PS, not
surface treated, and are therefore hydrophobic. Thirty-five-millimeter Petri dishes
are available from Greiner Bio-One GmbH, Frickenhausen, Germany, catalog num-
ber 627 102. Patterning will not work on tissue culture dishes. Patterning or uniform
modification of polymers other than PS is reported (Schu¨tte et al., 2010); however,
our previous studies indicate that PS substrates, being initially very cell repellent,
yield the best cell patterns. If Petri dishes cannot be used (some surface analysis tech-
niques require conductive substrates, cell cultures that can be combined with elec-
tronic interfaces, and so on), conventional silicon wafers, gold-coated silicon wafers,
or glass coverslips, can be spin coated with PS.
To improve the adhesion of PS onto Si wafers or glass, a layer of aminopropyl-
trimethoxysilane (APTMS [13822-56-5]) should be applied first. Aminopropyl-
triethoxysilane (APTES [919-30-2]) works equally fine. Both silanes react with
water and form thin, cross-linked layers on the surface of glass or Si wafers. Store
both compounds in tightly sealed bottles and use appropriate safety equipment.
3.1.5.1Protocol for spin coating of Si wafers or glass with PS films
Clean the substrate by boiling it for approximately 30 min in a solution of one part
per volume aqueous ammonia (30%), one part per volume hydrogen peroxide (30%),
and five parts distilled water. This solution has to be prepared just prior to use.
Rinse well with distilled water and blow dry with a nitrogen stream.
Prepare the silanization solution: This solution contains 94 volume parts metha-
nol, 5 volume parts 1 mM aqueous acetic acid, and 1 volume part APTMS or APTES.
The stock solution without the silane is stable; after adding the silane, hydrolysis will
start immediately, and the solution is therefore discarded after single use.
Incubate samples in the silanization solution for 2 h.
Rinse well with methanol or ethanol, apply an ultrasonic bath, and blow dry.
The silanization step can be omitted, but this will result in poor PS adhesion,
especially under cell culture conditions.
413.1Equipment and Protocols

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Micropatterning In Cell Biology Part A 1st Edition Matthieu Piel And Manuel Thry Eds

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