Field Work

Magnetic fields have done much for biology, from magnetic resonance imaging to micromanipulation of cells. Now, in an article in Nature Nanotechnology, Souza et al. report that magnets might replace rotational flasks or gel-based matrices for 3-D cell culture. As even the most cursory examination of any multicellular organism will show, the native environment of cells is one surrounded by other cells and structural features, so moving beyond traditional 2-D culture would enhance the fidelity of in vitro systems in mimicking the complex intercellular interactions and functional roles found in vivo. In Souza et al.'s strategy, the cells are made responsive to a magnetic field by incubating them with dispersed fragments of a hydrogel formed from gold nanoparticles, magnetic iron oxide nanoparticles, and phage particles displaying an integrin-binding peptide. The hydrogel fragments bind to the cell and can undergo integrin receptor-mediated internalization, so when a magnet is placed above the culture vessel, the cells are levitated within the medium. Since the magnetic force is not sufficient to overcome the surface tension of the medium, the cells accumulate at the liquid-air interface, where they gather directly under the magnet and form spherical multicellular structures. Compared to cells in 2-D culture, magnetically levitated human glioblastoma cells did not require passaging and grew faster. Based on expression of N-cadherin, a marker of intercellular contact, the levitated cells also more closely resembled cells growing in the in vivo model system of human tumor xenografts in immunodeficient mice. Preliminary studies also showed that the multicellular structure could be influenced by the shape of the magnet, while experiments in which separately cultured glioblastoma cells and astrocytes were later combined showed the feasibility of studying interactions of different cell types brought together by magnetic guidance. Compared with scaffolds made of biodegradable material, magnetic levitation offers simpler setup and much faster propagation. Moreover, the magnetic hydrogels can easily be adapted to incorporate phage bearing other peptide ligands and—unlike some 3-D culture methods—allow use of standard culture medium. The new tissue culture system is expected to have applications in bioengineering, stem cell research, and biotechnology.

Magnetic fields, such as illustrated above, are enabling 3-D culture of cells made magnetically responsive through incubation with iron oxide and gold nanoparticles. (Click to enlarge)

Souza et al. Three-dimensional tissue culture based on magnetic cell levitation. Nat. Nanotech. [Epub ahead of print, March 14, 2010; doi: 10.1038/NNANO.2010.23].

Make the Switch

Different flavors of fluorescent highlighter proteins respond to light by switching on, changing emission wavelength, or toggling their fluorescence on and off in response to particular illumination triggers. Unlike static versions, such as GFP, highlighters are perfect for pulse labeling, making them ideal for visualizing protein trafficking processes. Photo-convertible varieties of highlighters, which are fluorescent before and after a light trigger, are particularly popular because they simplify cell tracking. However, current photoconvertible fluorescent proteins can be hamstrung by inefficiency, rapid bleaching, or the need for multi-merization. In the Journal of Biological Chemistry, Welman et al. offer a new tool, photoactivatable Green Cherry (G^PAC), which is a fusion of the red fluorescent protein (RFP) monomeric Cherry with a photoactivatable variant of GFP. The fusion protein is designed to offer uninterrupted red fluorescence to designate the marker-expressing cells, while green fluorescence should remain nonexistent until triggered by a burst of 405-nm light. As intended, G^PAC-expressing cells showed strong red signal before and after photoactivation, and green fluorescence was trigger-dependent and persisted for a few hours. In several tagged proteins tested, G^PAC tolerated fusion proteins positioned at its N or C terminus without losing activity or noticeably affecting localization or function of the cellular protein, even when the fusion partner required extensive posttranslational processing and modification such as myristoylation or farnesylation. The authors show the application of G^PAC in investigating cytoskeleton dynamics in cultured cells, and in studying migration of immune cells in vivo in Drosophila. Such in vivo applications are likely to be a particular advantage of the G^PAC strategy, which—unlike the recently described photoconvertible protein Pharmet—does not rely on cyan fluorescence or fluorescence resonance energy transfer (FRET), both of which can suffer from limited range in tissue. One potential stumbling block for G^PAC is the relatively large size of the fluorescent tag (>50 kDa); this feature requires G^PAC-fused proteins to be carefully validated before using in functional studies. Nevertheless, G^PAC offers a convenient means to follow tag-expressing cells or subcellular compartments (via red fluorescence) while highlighting a photoconverted subset (green signal) in ongoing monitoring of spatiotemporal dynamics.

Welman et al. 2010. Two-color photo-activatable probe for selective tracking of proteins and cells. J. Biol. Chem. 285(15):11607–11616.