Cell culture is an essential tool for drug discovery, tissue engineering, and stem cell research. Conventional tissue culture produces two-dimensional (2D) cell growth with gene expression, signaling, and morphology that can differ from those in vivo and thus compromise clinical relevancy1–5. Here we report a three-dimensional (3D) culture of cells based on magnetic levitation in the presence of hydrogels containing gold and magnetic iron oxide (MIO) nanoparticles plus filamentous bacteriophage. This methodology allows for control of cell mass geometry and guided, multicellular clustering of different cell types in co-culture through spatial variance of the magnetic field. Moreover, magnetic levitation of human glioblastoma cells demonstrates similar protein expression profiles to those observed in human tumor xenografts. Taken together, these results suggest levitated 3D culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and allows for long-term multi-cellular studies.

Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d).

Biological molecular assemblies are excellent models for the development of nanoengineered systems with desirable biomedical properties. Here we report an approach for fabrication of spontaneous, biologically active molecular networks consisting of bacteriophage (phage) directly assembled with gold (Au) nanoparticles (termed Au-phage). We show that when the phage are engineered so that each phage particle displays a peptide, such networks preserve the cell surface receptor binding and internalization attributes of the displayed peptide. The spontaneous organization of these targeted networks can be manipulated further by incorporation of imidazole (Au-phage-imid), which induces changes in fractal structure and near-infrared optical properties. The networks can be used as labels for enhanced fluorescence and dark-field microscopy, surface-enhanced Raman scattering detection, and near-infrared photon-to-heat conversion. Together, the physical and biological features within these targeted networks offer convenient multifunctional integration within a single entity with potential for nanotechnology-based biomedical applications.target ͉ fractal ͉ hydrogel ͉ stem cell ͉ assembly I n nature, the assembly of molecules and particles is often directed by hydrophobic, van der Walls, and͞or electrostatic interactions (1, 2). Biological systems in particular are driven toward energetically favorable structures that have molecular selectivity and recognition and thus serve as models for nanoscale engineering-based molecular assembly. Given that filamentous bacteriophage (phage) (3-6) are resistant to harsh conditions such as high salt concentration, acidic pH, chaotropic agents, and prolonged storage (6), they are suitable candidate building blocks to meet the challenges of bottom-up nanofabrication (1, 2). Moreover, the pIII minor capsid protein of the phage can be easily engineered genetically to display ligand peptides that will bind to and modify the behavior of target cells in selected tissues (3,4,(6)(7)(8). Thus, the tactic of integrating phage display technology with tailored nanoparticle assembly processes offers opportunities for reaching specific nanoengineering and biomedical goals (1-4, 6, 8-10).In this work, we show that such networks are biocompatible and preserve the cell-targeting and internalization attributes mediated by a displayed peptide and that spontaneous organization (without genetic manipulation of the pVIII major capsid protein), and optical properties can be manipulated by changing assembly conditions. By taking advantage of gold (Au) optical properties (11-16), we generated Au-phage networks that, in addition to targeting cells, can function as signal reporters for fluorescence and dark-field microscopy and near-infrared (NIR) surface-enhanced Raman scattering (SERS) spectroscopy. Notably, this strategy maintains the low-cost, high-yield production of complex polymer units (phage) in host bacteria and bypasses many of the challenges in developing cell͞peptide detection tools, such as complex syn...