TECHNOLOGY FEATURE

Scratching the surface 589 Moving in a 3D world 590 An image worth a thousand words 592 Box 1: A deadly difference 590

Cell migration: our protruding knowledge Nathan Blow looks at some of the technologies used by cell biologists to unlock the mysteries of

methods cell migration.

As people move from place to place of cells and chemoattractants. Today, to carry out daily activities, with- Neuroprobe is taking the Blind Well .com/nature e in their bodies cells also move to chamber into the high-throughput perform many critical biological world with 96- and 384-well dispos- .natur functions. Development, immune able ChemoTx systems and a 1,536- w response and wound healing all rely well variant in development. The on the directed migration of cells. use of a single membrane covering When cell migration goes awry, all the wells eliminates the need to http://ww

the effects can be very bad, indeed, handle many small, fragile mem-

oup ranging from tumor metastasis to branes during the assay. r

G vascular diseases.

Cell migration involves com- Scratching the surface plex protein signaling cascades and Another classic, in the low-tech lishing changes in the structure of the cell’s category, is the ‘wound healing’ b actinomyosin cytoskeleton. Thus assay, which is used to study migra- Pu

Ibidi is developing slides for long-term studies of cell migration. biologists studying cell migration (Courtesy of Ibidi GmbH.) tion of cells in the absence of a wear many hats in the laboratory, chemoattractant. A confluent plate

Nature and embrace new methodologies, of cells is ‘wounded’ by scraping a

7 technologies and even dimensions in migration, including chemotaxis, although specific area of the plate. Migration of

200 the quest to discover how and why cells the original design has been modified and cells can then be monitored over time by

© move. improved upon since the system was first imaging the cells as they move from the described in 1962. Separating the two untouched areas into the wounded area. New twists on one of the classics chambers is a porous membrane through Although it is simple, this assay is also Although some cell movements are ran- which cells can actively migrate. The size cumbersome because imaging cells over dom, others are directed toward a signal. of the membrane pores is critical when long periods can be difficult, and at first One such process, chemotaxis—which using a Boyden chamber, and companies glance the system does not appear to be occurs in response to a chemical gradi- sell membranes with a variety of pore amenable to automation. ent—has been extensively studied. A clas- sizes. A typical eukaryotic cell is 30–50 µM Enter Applied BioPhysics, which has sical way to study chemotaxis is to use a in size and can fit through pores in the 3– developed a wound healing assay based two-chamber system: cells are placed in 13-µM size range, but various companies on the concept of electric cell-substrate one chamber and the chemical signal, or have optimized pore size for specific cell impedance sensing (ECIS). One small chemoattractant, in the other. types. For example, Millipore supplies the electrode is placed on the bottom of a Several variations of two chamber sys- QCM Quantitative Cell Migration assay tissue culture well in the center, and a tems have been used including a top-and- that uses an 8-µM pore size membrane, second electrode at the edge of the well bottom configuration known as the Boyden optimal for epithelial and fibroblast cell completes the circuit. Cells that attach to chamber, a side-by-side chamber configu- migration assays. the electrode in the culture well alter the ration known as a Zigmond chamber and The company Neuro Probe, founded current, and this effect can be measured the Dunn chamber, which consists of con- in 1970, has based a large part of its busi- directly. “Cells are basically insulating, so centric rings. More recently, Ibidi intro- ness on improving the Boyden chamber. when you put the cells on the electrode, duced the µ-Slide chemotaxis assay, which “We simplified the design of the Boyden the current has more difficulty flow- allows real-time imaging of chemotaxis on chamber for use and production, and fur- ing, so the resistance and the impedance a microscope slide. ther refined the design—creating the Blind increase,” says Ivar Giaever, president of The Boyden chamber was the first two- Well chamber,” says Mark Phillips, presi- Applied BioPhysics. By growing the cells chamber system to be described1. It is still dent of Neuro Probe, adding that its main to confluence and then applying an elec- used today to study many types of cell advantage is the smaller required volume tric field for 10–15 seconds, one kills the

NATURE METHODS | VOL.4 NO.7 | JULY 2007 | 589 TECHNOLOGY FEATURE

cells on the electrode. As the surrounding migration are moderately higher,” he says cells migrate into the wounded area, the of the 3D culture experiments, “but what associated change in current can be mea- was striking was the cells were migrating sured to calculate the cell migration rate. more directionally, and that was not relat- The advantage of a system like this is ed to an intrinsic pattern in the matrix”. clear: it can be automated. “You can grow Yamada’s group and others have since cells in tissue culture and then say, ‘I want shown that it the matrix structure itself to wound them at midnight’. The system is likely responsible of the effect2. “It is will do that, and you get all the data when something about the three-dimension- you get back in the morning,” says Giaever. ality, not a particular component,” says Applied BioPhysics also offers this assay in Yamada. He points to work by Edna a 96-well culture dish format, providing a Cukierman in his lab and the work of methods Human umbilical vein endothelial cells (HUVEC) high-throughput solution. other groups suggesting that the stiff- cultured in the 3D PuraMatrix Synthetic ness of the extracellular matrix might be Nanofiber Scaffolds and stained with rhodamine- Moving in a 3D world important in directed migration, and that phalloidin (red) and DAPI (blue). (Courtesy of The classical tissue culture dish is a two- making a matrix stiffer makes cells behave Alisha Sieminski, Olin College.) .com/nature e dimensional (2D) environment in which more like in two dimensions. cells lack the components and structure of Yamada thinks that the matrix compo- of home-brewed matrices, and commer- .natur the three-dimensional (3D) extracellular sition and compliance are important in cial companies now also supply matrices w matrix they would see in vivo, composed the ability of cells to migrate and there- for cell culture studies. These matrices can of collagens, proteins and proteoglycans. fore needs to be considered when design- be either synthetic or derived from animal Migration of some cell types differs ing experiments. “In fact, it is pretty clear products, allowing researchers to tailor an http://ww

dramatically between 2D and 3D worlds that 3D matrices are different in different extracellular matrix to their needs.

oup (see also Box 1). Kenneth Yamada at the tissues and that you will need to match, in PuraMatrix Synthetic Nanofiber r

G US National Institutes of Health realized terms of stiffness and composition, your Scaffolds from 3DM Inc. are amino

this quickly after analyzing his group’s particular tissue.” acid–based hydrogel scaffolds suitable for early results studying migration of human To help understand cells in 3D environ- 3D cell culture. Developed by Shuguang lishing fibroblasts in three dimensions. “Rates of ments, researchers have made up a variety Zhang from the Massachusetts Institute of b Technology, PuraMatrix is based on 16- Pu mer oligopeptides that self-assemble into BOX 1 A DEADLY DIFFERENCE nanofibers that are 10–50 nm in diameter,

Nature to form a synthetic extracellular matrix

7 “Migration and invasion are related, but they are not the same,” says Mina Bissell at physiological salt concentrations. from Lawrence Berkley National Laboratory. “You cannot invade without migrating, 200 PuraMatrix can mimic many aspects of

© but you could migrate without invasion.” The two processes also can have the 3D cellular environment, without the dramatically different outcomes. need for animal materials. “PuraMatrix Bissell’s recent work focuses on characterizing the invasion properties of malignant is customizable. You can change the bio- cells in three dimensions, properties that allow these cells to break through the physical or biochemical characteristics of basement membrane and enter the bloodstream. This process relies on proteins such the material by either altering the concen- as matrix metalloproteases to ‘chew’ through the membrane. “We needed a new assay tration of the matrix, which alters stiffness for invasion, that includes migration, to explain how normal cells invade but respect and porosity, or by adding in components tissue boundaries, whereas tumor cells invade and destroy tissue structure,” says such as extracellular matrix proteins or Bissell. growth factors,” says Lisa Spirio, direc- Celeste Nelson, a bioengineering fellow in Bissell’s laboratory, developed a tor of research and development at 3DM. quantitative assay using micropatterns of cells sandwiched between two layers of Additionally, this matrix is transparent, collagen. The model, recently described in Science4, is based on the process involved allowing visualization of cell movement. when the mammary gland invades the fat pad during puberty, a normally very well controlled process. Once cells become malignant, in contrast, this tight spatial “Some of the harder scaffolds or mem- control is lost, and the cells invade beyond the fat pad. This gave Bissell an idea for a brane filters are opaque, which is a chal- way to study invasion and tumor-cell metastasis. “If we understood the invasion and lenge for microscopy and analysis,” says control of the cells into the fat pad, then we could find out how it is that tumor cells Zen Chu, president of 3DM. hijack this pathway.” Another matrix that has found wide use Using their model, Nelson, Bissell and her group showed that the tissue in the scientific community is Matrigel architecture surrounding the cell has critical effects on mammary cell branching into from BD Biosciences. Matrigel is a solu- the mammary gland structure, and how these cells migrate and invade collagen. bilized basement membrane preparation Bissell insists that the difference between invasion and migration needs to be extracted from the Engelbreth-Holm- understood and made clear. As Bissell points out, “if you have a wound in your skin, Swarm (EHS) mouse sarcoma cells. At the fibroblasts migrate but they don’t invade, because if they are able to invade, room temperature, Matrigel polymerizes sooner or later, you will end up with a tumor!” into a three dimensional structure that is useful for both cell culture and studying

590 | VOL.4 NO.7 | JULY 2007 | NATURE METHODS TECHNOLOGY FEATURE

cellular processes in three dimensions, discrimination of things that are at the including cell migration. The rigidity of membrane surfaces,” says Jennifer Peters the matrix formed by Matrigel can be from Nikon Instruments, which offers the varied, allowing user-defined stiffness to TIRF-2 illuminator system. more closely resemble natural microenvi- Among the most popular combination ronments. of labeling method and microscopy sys- Trevigen supplies its Cultrex prod- tem is speckle microscopy. This visual- ucts, three matrix formulations that vary ization method was established by Clare according to the concentration of base- Waterman-Storer of the Scripps Research ment membrane. Derived from EHS Institute, with the help of a small dose mouse sarcoma cells, like Matrigel, the of serendipity, as her Scripps colleague Cultrex matrices can be used for tumor- Gaudenz Danuser recalls. “Clare wanted methods genicity, migration, growth, differentia- to label actin in a continuous mode, and tion and neurite-outgrowth assays. The she injected a pool of labeled mono- ECMatrix from Millipore, which is used mers, which was really, really poorly in the company’s cell migration kits, is labeled, so only a few of these monomers .com/nature e also a reconstituted basement membrane had a label at all,” says Danuser. When matrix of proteins derived from EHS Waterman-Storer cranked up the camera .natur mouse tumor cells. amplification, a few labeled molecules w could be seen. But interestingly, these An image worth a thousand words labeled molecules showed movement and Another important challenge in studying could be tracked. This gave Danuser and http://ww

cell migration is to understand what hap- Waterman-Storer the idea to use speckle

oup pens in a cell when it moves. Imaging the microscopy to examine molecule move- r

G cytoskeleton proteins, for example, dur- ment in cells.

ing cell migration on 2D surfaces requires Although they could tell the speckles some form of labeling in addition to moved, Danuser says it took over six years lishing specialized microscopy such as confocal to develop the necessary computational b and total internal reflection fluorescence algorithms to quantitatively track their Pu (TIRF) systems. movements. “We have developed over the TIRF seems to be the microscopy method past six years a comprehensive mathemat-

Nature of choice for studying interactions and ical model that allows you to take the sto-

7 structures at the edges of cells. The great chastic fluctuations of the speckles, track

200 advantage of TIRF for this purpose is that their motion, interpret the motion, enter

© the illumination does not penetrate very mechanical events in the cell and also deep into the cell. Therefore fluorophores measure the rates of assembly and disas- inside the cell do not contribute to the sig- sembly of your structure, in situ.” nal, and the background tends to be very Recently, Danuser and Waterman-Storer low. Overall, using TIRF “you get good applied speckle microscopy to better

Nikon provides TIRF-based imaging systems. (Courtesy of Nikon Instruments.)

592 | VOL.4 NO.7 | JULY 2007 | NATURE METHODS TECHNOLOGY FEATURE

limitation of speckle microscopy is that we are limited to processes that happen at a time scale of half a second or slower, while methods like fluorescent correlation spec- trometry can detect binding of molecules at a millisecond time scale,” says Danuser. The cause of this is mainly technical as Danuser explains. “Signals are extreme- ly low and the [charge-coupled device (CCD)] camera needs a certain amount of time to integrate enough photons.” Cell biologists have come long way in methods the study of cell migration. With advances being made on many fronts, it is only a 0:04:00 0:07:30 0:11:00 0:14:30 matter of time until cell migration really comes into focus. .com/nature Time lapse speckle microscopy images of actin (red) and vinculin (green). (Courtesy of the Waterman- e Storer lab.) 1. Boyden, S. J. Exp. Med. 115, 453–466 (1962). 2. Pankov, R. et al. J. Cell. Biol. 170, 739–802 .natur w (2005). understand the proteins that interact with movements of these molecules during cell 3. Hu, K. et al. Science 315, 111–115 (2007). actin during cell movement3. By labeling migration. 4. Nelson, C.M. Science 314, 298–300 (2006). proteins with GFP, labeling actin mono- So it turns out that speckle microscopy http://ww

mer with a different dye and using TIRF is an excellent tool for studying the mole- Nathan Blow is technology editor

oup microscopy and software they had devel- cules involved in cell migration, but the for Nature and Nature Methods r

G oped, they could study interactions and technique does have drawbacks. “The ([email protected]). lishing b Pu

Nature

7 200 ©

NATURE METHODS | VOL.4 NO.7 | JULY 2007 | 593 TECHNOLOGY FEATURE

SUPPLIERS GUIDE: COMPANIES OFFERING SYSTEMS AND REAGENTS TO STUDY CELL MIGRATION Company Web address 3DM http://www.puramatrix.com Accuri Cytometers http://www.accuricytometers.com Agilent http://www.agilent.com Andor Technology http://www.andor-tech.com Apogee Instruments http://www.ccd.com Applied BioPhysics http://www.biophysics.com Applied Cytometry http://www.appliedcytometry.com Applied Scientific http://www.asiimaging.com

methods BectonDickinson http://www.bd.com Beckman Coulter http://www.beckmancoulter.com BioCat GmbH http://www.biocat.de Biomedical Photometrics http://www.genefocus.com

.com/nature Bio-Rad http://www.bio-rad.com e Cambio http://www.cambio.co.uk

.natur Cell Biolabs Inc. http://www.cellbiolabs.com w Chroma Technology http://www.chroma.com Clontech http://www.clontech.com EMD Biosciences http://www.emdbiosciences.com http://ww

Eppendorf http://www.eppendorf.com

oup Fujifilm http://www.fujimed.com r G

Guava Technologies http://www.guavatechnologies.com Harvard Apparatus http://www.harvardapparatus.com Inbiolabs http://www.inbiolabs.com lishing b Innovative Cell Technologies http://www.innovativecelltech.com Pu

Invitrogen http://www.invitrogen.com Ibidi http://www.ibidi.com Kodak http://www.kodak.com Nature

7 La Vision Biotec http://www.lavisionbiotec.com Leica Microsystems http://www.leicamicrosystems.com 200

© Lightools http://www.lightools.com Micro Video Instruments http://www.mvi-inc.com Millipore http://www.millipore.com Molecular Devices http://www.moleculardevices.com MP Biomedicals http://www.mpbio.com Nanoptek http://www.nanoptek.com Neuroprobe http://www.neuroprobe.com Nikon Instruments http://www.nikon-instruments.jp/eng/ Olympus http://www.olympus.de/microscopy Omega Optical http://www.omegafilters.com Perkin Elmer Life Sciences http://las.perkinelmer.com Pierce Biotechnology http://www.piercenet.com Princeton Instruments http://www.princetoninstruments.com Qimaging http://www.qimaging.com Trevigen http://www.trevigen.com Sigma Aldrich http://sigmaaldrich.com Synthecon http://www.sythecon.com Zeiss http://www.zeiss.com

594 | VOL.4 NO.7 | JULY 2007 | NATURE METHODS