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.