Abstract

Engineering of the cellular microenvironment has become a valuable means to guide cellular activities such as spreading, motility, differentiation, proliferation, or apoptosis. This chapter summarizes recent approaches to surface patterning METHODS IN CELL BIOLOGY, VOL. 83 Copyright 2007, Elsevier Inc. All rights reserved. 0091-679X 07 35.00 DOI 10.1016 S0091-679X(07)83005-6 such as topography and chemical patterning from the micrometer to the nanometer scale, and illustrates their application...

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Fig. 4 Local micromechanics of single cells embedded in a 3D matrix. (A) Fluorescent micrograph of the actin filament architecture in a human umbilical venous endothelial cell (HUVEC) embedded in a 3D puramatrix gel, overlaid with a fluorescent micrograph of nanoparticles embedded in the cytoplasm. Large color-coded dots were drawn over each nanoparticle to aid presentation. Blue denotes the least deformable (stiffest) regions of the cell red denotes the most deformable (softest) regions. Actin...

Atomic Force Microscopy

Several key aspects of the microsphere indentation technique are implemented and extended in a commercially available, high-resolution instrument known as the AFM. A number of designs are available from different AFM manufacturers, with prices starting at about half the cost of an inverted optical microscope. While imaging applications of AFM are relatively well known (Bennig et al., 1986), the AFM in the force mode'' is highly suited to detailed mechanical assessments at microscale resolution...

B

Fig. 3 Particle-tracking nanorheology of cells manual injection versus ballistic injection of nanopar-ticles. (A) Phase contrast micrograph of Swiss 3T3 fibroblasts overlayed with a fluorescent micrograph of nanoparticles that were ballistically injected into their cytoplasm. (B) Comparison between the mean cellular compliance computed from the displacements of nanoparticles microinjected (blue) (n 3) and bombarded (red) (n 30) into the cytoplasm of Swiss 3T3 fibroblasts. Figures reprinted with...

Conclusions

With the realization of the importance of mechanical signals, studies of cell biology are increasingly performed on polymer-based substrates with tunable mechanical properties in an effort to elucidate the mechanisms that regulate the complex cell behavior. The quality of these studies is directly affected by the proper characterization of the mechanical properties of these substrates. While AFM represents a highly reliable and versatile approach, it is also the most costly and complicated...

Na

MEF deficient in lamin A C (Lmna- - MEF) HUVEC cell on a peptide 2D matrix treated with VEGFk HUVEC inside a peptide 3D matrix treated with VEGFk Interphase nucleus of Swiss 3T3 fibroblast1 Particle-tracking nanorheology was used to study the mechanical properties of many varieties of cells under a wide range of conditions. Unless stated, all values of viscosity and elasticity in the table are for the cytoplasm. The elasticity was evaluated at a shear frequency of 1 sec-1 (1 sec-1 1 Hz) and the...

And Alternative Uses

Georges,* Paul A. Janmey,* and Karen A. Beningo* Department ofBiology Wayne State University Detroit, Michigan 48202 Department of Bioengineering Institute for Medicine and Engineering University of Pennsylvania Philadelphia, Pennsylvania 19104 II. Principle of the Polyacrylamide Hydrogel A. Standard Method of Polymerization B. Light-Induced Initiation of Polymerization III. Conjugation of Proteins to Polyacrylamide A. Carbodiimide-Mediated Cross-Linking B....

A

Available Software Packages and Computing Resources Source code for our IDL particle-tracking routines is available for free download at along with a short online tutorial. These particle-tracking routines have been translated into the increasingly popular MatLab environment. Both IDL and MatLab are available for a wide variety of computing platforms and many research institutions have a site license for at least one of them. Moreover, a stand-alone version (with a graphical user interface)...

Pa

Unstable reactive O-acylisourea ester Fig. 4 The carbodiimide method of protein coupling. EDC reacts with acrylic acid incorporated into the polyacrylamide and activates the carboxyl group to form an unstable reactive O-acylisourea ester intermediate. This reaction allows it to be coupled to primary amines. that drives the polymerization of polyacrylamide (Menter, 2000). A final concentration of 0.2 acrylic acid, which should be adjusted in proportion to the concentration of total acrylamide,...

Info

Where a is the opening angle of the pyramid tip (i.e., how tapered the tip is other symbols are the same as defined before). Blunted tips typically have an opening angle, a, of 35whereas sharpened tips have an angle of 18 . Note that the smaller contact area with the pyramidal tip relative to a spherical tip means that the indentation force scales more strongly with deformation, d (as d2 rather than d3 2). The difference gives spherical probes an advantage, as it translates to lower strains...

Introduction

The interaction between a cell and its surrounding matrix is important for cell adhesion, migration, and differentiation. Our current understanding of this interaction is largely derived from studies of cells on flat tissue culture dishes made either with synthetic polymers such as polystyrene (PS) or glass coverslips coated with extracellular matrix proteins, like collagen or fibronectin. However, compared to these flat surfaces, tissues present a three-dimensional (3D), more textured...

Cd 400

-30 -20 -10 0 10 20 30 Deformation ( ) Fig. 14 Effects of compression or elongational deformation on shear modulus of intact rat brain tissue under 2 oscillatory shear strain. Compression of brain tissue (negative deformation) increases the shear modulus, and at 20 compression the shear modulus G has increased by nearly a factor of 3. as the arterial wall (Shadwick, 1999). The PP geometry will exaggerate strain stiffening since most of the stress is carried at the edge of the sample, and...

Pdms

Annealed at 150 C for 10 h Reactive ion etching to oxidize PS Fig. 2 The patterns formed by electrodynamic instability can be used as a master template for lithography to produce substrates of oxidized PS. Inc.) for 1 day in a vacuum oven at 150 C. After peeling the PDMS from the PS, a PS film with posts that match those formed in the original PMMA film was formed (Fig. 2B). The surface of the PS can be oxidized using a commercially available reactive ion etcher (oxygen ion at 25 W for 30 sec,...

Patterning at the Micro and Nanoscale with Polymer Mixtures and Block Copolymers

Many techniques are available to fabricate patterned surfaces, but these methods are limited by the size of the patterned area and or minimum feature size of 100 nm that can be routinely and reproducibly achieved. One strategy to overcome this limitation relies on the morphology generated by the microphase separation of diblock copolymers (explained below), the self-assembly of a mixture of homopolymer blend and diblock copolymer, and the macroscopic phase separation of homopolymer blends to...

Introduction Sensing Cellular Environments

Cell adhesion to the extracellular matrix (ECM) and to neighboring cells is a complex, tightly regulated process that plays a crucial role in fundamental cellular functions, including cell migration, proliferation, differentiation, and apoptosis (Blau and Baltimore, 1991 Ruoslahti and Obrink, 1996). By interacting directly with such external surfaces, cells gather information about the chemical and physical nature of the ECM, integrate and interpret it, and then generate an appropriate...

Probing Intracellular Force Distributions by High Resolution Live Cell Imaging and Inverse Dynamics

LinJi,1 Dinah Loerke,1 Margaret Gardel, and Gaudenz Danuser The Scripps Research Institute LaJolla, California 92037 A. Actin Cytoskeleton Mechanics in Cell Protrusion C. Probing Heterogeneous Network Elasticity with Speckle Microscopy A. Solution of the Inverse Problem References Highly coordinated molecular regulation of mechanical processes is central to numerous cell processes. A key challenge in cell biophysics is, therefore, to probe intracellular force distributions and mechanical...

Imaging Stress Propagation in the Cytoplasm of a Living Cell

Ning Wang,* Shaohua Hu,* and James P. Butler* Department of Mechanical Science and Engineering University of Illinois at Urbana-Champaign Urbana, Illinois 61801 Physiology Program Harvard School of Public Health Boston, Massachusetts 02115 II. Detecting External Stress-Induced Displacements in the Cytoplasm A. Green Fluorescent Protein Transfection and Cell Culture B. Application of Periodic Mechanical Stress III. Imaging Displacement and Stress Maps in a Live Cell A. Quantifying Displacement...

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The relaxation modulus contains information about how rapidly structures can reorganize to relieve the stress in the system. A rheometer is any instrument that enables determination ofrheological properties. Very simple rheometers such as capillaries have been used for centuries to determine flow of liquids, and in the art of cooking people have used simple kitchen tools or teeth and fingers as a means to probe rheological properties of food products. Modern rheometers allow a more precise...

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Produces a chromophore by-product that can be quantified by spectrophotometry, thus providing information about the amount of ligand actually bound to the gel. This technique provides several advantages over the original design. First, it incorporates the protein-coupling reagent throughout the gel, eliminating the issue of uniform distribution and allowing rapid reaction with amine groups. Second, it addresses a weakness in the initial design of the polyacrylamide hydrogels an accurate method...

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J LJ-LJ 4 -0.5 0.0 0.5 1.0 1.5 Ax (pixels) x mod 1 (pixels) Fig. 6 (A) Tracer trajectories from the region of interest. (B) Histogram of tracer displacements between consecutive frames, which decays well before 4 pixels, the maximum displacement set in the particle-tracking software. (C) Histogram of the x-position of the particle modulo 1, demonstrating that there is negligible systematic error with these choices of tracking parameters.

Mbw

(A x2(t) ((x(t + t)-x(O)2 (3) where x indicates tracer position in one dimension, t is time, and < > indicates time and ensemble (over all particles) average. However, it may be best understood graphically. Figure 7B shows a histogram of the distance of travel from 200 particles diffusing in a liquid for 0.02 and 0.4 sec, respectively. The MSD at given lag time is just the square of the standard deviation of particle positions after a given lag time. To convert a one-dimensional MSD,...

Irene Y Tsai Alfred J Crosby and Thomas P Russell

Polymer Science and Engineering Department University of Massachusetts Amherst, Massachusetts 01003 II. Patterning with Electrodynamic Instabilities A. Procedure for Patterning with Electrohydrodynamic Instabilities B. Cell Migration on Topographic Surfaces III. Lithography Without a Clean Room IV. Patterning at the Micro- and Nanoscale with Polymer Mixtures and Block Copolymers A. Principles and Procedures for Controlling Pattern Formation with Polymer Mixtures and Block Copolymers V. Summary...

WYh

Fig. 2 Elasticity and plasticity are relevant on different timescales. (A) In an elastic spring system, Hooke's law states that F ku L, with u the elongation, L the resting length, k the elastic spring constant, and F the applied force. When spring constant and resting length are known, the applied force can be calculated from the observed elongation. (B) In an actin network (represented as a system of springs), the elastic properties can change over time due to dynamic remodeling at long...

Multiple Particle Tracking and Two Point Microrheology in Cells

Hoffman Department of Chemical and Biomolecular Engineering Institute for Medicine and Engineering University of Pennsylvania Philadelphia, Pennsylvania 19104 II. Principles of Passive Tracer Microrheology A. Conventional Passive Microrheology B. Expected Tracer Motion and Tracking Performance III. Multiple-Particle Tracking Algorithms B. Locating Possible Particle Positions C. Eliminating Spurious or Unwanted Particle Trajectories D. Linking Positions into...

G

Body-centered cubic arrays of spheres, hexagonally packed cylinders, bicontinuous gyroids, or alternating lamellae (Fig. 5) (Bates and Fredrickson, 1990,1999 Hamley, 2004 Hashimoto et al., 1983). The architectures of block copolymers can be expanded further to multiblock copolymers, for example triblock or pentablock, star- or comb-shaped block copolymers, where the chemical composition of the blocks can be two, three, or more chemically different polymers. In addition, rather then linking...

Preface

Most eukaryotic cells possess a well-recognized sense of taste or smell'' mediated by diffusible factors, which bind to cellular receptors and initiate signaling cascades. However, cells also possess an oft-overlooked sense of touch. This sense is rooted in cell mechanics and dependent on various cytoskeletal structures and processes that detect, modify, and respond to physical parameters of the environment. In particular, many types of cells are found to respond to mechanical force and...

Contents

PART I Basic Concept and Preparation Culture Substrates for Cell Mechanical Studies 1. Basic Rheology for Biologists Paul A. Janmey, Penelope C. Georges, and Soren Hvidt I. Introduction and Rationale 4 III. Rheological Instrumentation 11 IV. Experimental Design 13 V. Sample Preparation 20 VI. Special Considerations for Biological Samples 22 2. Polyacrylamide Hydrogels for Cell Mechanics Steps Toward Optimization Casey E. Kandow, Penelope C. Georges, Paul A. Janmey, and Karen A. Beningo II....

Joachim P Spatz and Benjamin Geiger

Department of New Materials and Biosystems Max Planck Institute for Metals Research Stuttgart, Germany and Department of Biophysical Chemistry University of Heidelberg, Heidelberg, Germany Department of Molecular Cell Biology Weizmann Institute of Science Rehovot 76100, Israel I. Introduction Sensing Cellular Environments II. Nano-Digital Chemical Surfaces for Regulating Transmembrane Receptor Clustering A. Extended Nanopatterns and Biofunctionalization B. Micro-Nanopatterns for Spatially...

Drr R tAiTD2T4

Where Ar1 (t) and Ar2(t) are the stochastic motions of the first and second particle, respectively, along their mutual line of centers in lag time t (Fig. 8). The brackets indicate an average over all pairs of particles in the data set with separation R. In practice pairs with similar R values are pooled together to create a function of both R and t. Since the TPM signal's origin is Brownian fluctuations of the material, the t dependence should be related to the rheology in a manner resembling...

Chapter

Microscopic Methods for Measuring the Elasticity of Gel Substrates for Cell Culture Microspheres, Microindenters, and Atomic Force Microscopy Margo T. Frey,* Adam Engler,* Dennis E. Discher,* Juliet Lee,* and Yu-Li Wang* University of Massachusetts Medical School Department of Chemical and Biomolecular Engineering University of Pennsylvania Philadelphia, Pennsylvania 19104 Department of Molecular and Cell Biology University of Connecticut Storrs, Connecticut 06269 II. Probing with Microspheres...

Basic Rheology for Biologists

Georges,* and Soren Hvidt* Department of Bioengineering Institute for Medicine and Engineering University of Pennsylvania Philadelphia, Pennsylvania 19104 Departments of Physiology and Physics Institute for Medicine and Engineering University of Pennsylvania Philadelphia, Pennsylvania 19104 Department of Chemistry Roskilde University Roskilde DK-4000, Denmark Abstract I. Introduction and Rationale II. Rheological Concepts III. Rheological Instrumentation F. Strain...

D

Error for large indentations at least up to air 0.8, where a2 dr and a is the radius of the contact area, or d 0.64r for materials with a high Poisson's ratio (0.40 Yoffe, 1984). For the present measurements, Yoffe's correction for a material of v 0.40 amounts to a difference of less than 2 in the value of E. These results suggest that indentations up to the radius of the microindenter may be applied in the present approach in conjunction with an uncorrected Hertz model, for elastic materials...

Probing with Spherically Tipped Glass Microindenters

To address some of the limitations of microsphere indentation and AFM, a new approach was recently developed that uses a flexible glass microneedle with a spherical tip to probe the gel. The method is identical to AFM in principle, measuring the indentation of the gel in response to calibrated forces exerted at a spherical tip. However, the position of the probe and the indentation of the material are measured through an optical microscope with a calibrated focusing mechanism in conjunction...

Concluding Remarks

It was the objective of this chapter to collect a number of recently published improvements and alternative uses of the polyacrylamide hydrogel system in one place for evaluation by potential users. These improvements address the ultimate goal of using elastic substrates for the study of cellular mechanical interactions in a physiological relevant setting (Chapter 22 by Engler et al., this volume for relation to tissue elasticity). The improvements in protein coupling (Balaban et al., 2001...

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Fig. 3 The heterobifunctional photoreactive reagent sulfo-SANPAH. When the nitrophenyl azide reactive group is exposed to UV light (320-350 nm), it forms a nitrene group that can react nonspecifi-cally with polyacrylamide. The sulfosuccinimydyl group at the other end reacts with primary amine groups of the protein. can result in variations in the effectiveness of cross-linking. In addition, it is an expensive reagent with a limited shelf life. To overcome these shortcomings, a number of...

M

After polymerization remove top coverslip Fig. 2 The initial design of polyacrylamide hydrogels for cell culture. An activated acrylamide solution is dropped onto a coverslip with reactive surface that binds polyacrylamide. A second, nonreactive coverslip is then overlaid onto the acrylamide droplet. Following polymerization, the top coverslip is carefully removed. The hydrogel is then treated with the UV-activated heterobifunctional cross-linker sulfo-SANPAH for the covalent linkage of...

Summary

We have described several tools to study adhesion and migration of cells on 3D topographic substrates, which mimicks cells in physiological 3D environments. These techniques can be easily transferred to biological setting and applied to many types of cells such as epithelium, osteoblasts, and hepatocyte, during their differentiation and signal transduction. The method can be modified to pattern extracellular matrix proteins instead of synthetic polymers. In addition, such a substrates can be...

Volume

University of Massachusetts Medical School Department of Chemical and Biomolecular Engineering University of Pennsylvania Philadelphia, Pennsylvania AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN FRANCISCO SINGAPORE SYDNEY TOKYO Academic Press is an imprint of Elsevier Cover Photo Credit Schematic representation of intracellular forces at the leading edge of migrating adherent cells. Image created by James Lim, The Scripps Research Institute. From Chapter 9, Figure 1....

Probing with Microspheres Under Gravitational Forces

The microsphere indentation method is performed by measuring the indentation depth of a relatively heavy metal microsphere placed on top of the substrate (Lo et al., 2000). It works with a high degree of reproducibility for both polyacryl-amide and gelatin substrates with Young's moduli of E 2.2-33 kPa. Hydrogel substrates are prepared with embedded fluorescent latex beads 0.1-0.2 mm in diameter. As the method relies strongly on the ability to focus accurately on the substrate surface, using...

Probing Cellular Mechanical Responses to Stimuli Using Ballistic Intracellular Nanorheology

Lee,* Brian R. Daniels,* Thomas P. Kole,* Yiider Tseng,*'* and Denis Wirtz*'*' Department of Chemical and Biomolecular Engineering The Johns Hopkins University Baltimore, Maryland 21218 Department of Chemical Engineering University of Florida, Gainesville, Florida 32611 Department of Materials Science and Engineering The Johns Hopkins University Baltimore, Maryland 21218 Howard Hughes Medical Institute Graduate Training Program and Institute for...

Ax2t2 2RDrr

If the material is homogeneous and isotropic on length scales significantly smaller than the tracer, incompressible, and connected to the tracers by uniform no-slip boundary conditions over their entire surfaces, the two MSDs will be equal Ax2 t gt 2 Ax2 t gt . If these boundary and homogeneity conditions are not satisfied, the two MSDs will be unequal. In this case, applying the two-point MSD in the GSER will still yield the bulk rheology of the material on the long length scale R , while the...