Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue.

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.

The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules~LB 1-2 ! folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB 1-2 has now been solved using two-dimensional 1 H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and Ha chemical shifts and the low f and c angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB 1-2 structures superimposed well over LB 1 or LB 2 , but not over both modules simultaneously. LB 1 and LB 2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.Keywords: concatemer; LDL receptor; ligand-binding domain; NMR spectroscopy; protein structureThe low-density lipoprotein receptor~LDLR! plays a pivotal role in the removal of cholesterol-rich lipoproteins from the circulatioñ Havel & Kane, 1995!. LDLR binds to its ligands, apolipoproteiñ apo! B-100 and apoE, of LDL and intermediate-density lipoproteins, promoting their uptake by receptor-mediated endocytosis. Upon uptake, the receptor-ligand complex transits in endosomes, where the receptor separates from its ligand, and recycles back to the cell surface~Goldstein et al., 1985;Brown & Goldstein, 1986;Johnson et al., 1997!. The LDLR consists of ligand binding~LB!, epidermal growth factor~EGF! precursor homology, O-linked sugar, transmembrane, and cytoplasmic domains. The arrangement of these domains is similar to that of other members of the LDLR gene family, including the VLDL receptor~Takahashi et al., 1992!, the LDL receptorrelated protein~LRP!~Herz et al., 1988!, and the renal glycoprotein gp3300megalin~Saito et al., 1994!. The ligand-binding domain consists of seven imperfect repeats, each of 40-45 residues. The three-dimensional~3D! structures of the two N-terminal repeats, LB 1 and LB 2 , have been solved by NMR spectroscopy~Daly et al., 1995a, 1995b!, and LB 5 by X-ray crystallography~Fass et al., 1997!. Approximately 40% of the residues are conserved across the seven modules, including all cysteine residues, an isoleucine, and the D-x-S-D-E motif~Fig. 1!. The cysteine residues, which have a conserved I-III, II-V, and IV-VI pattern~Bieri et al., 1995a!, stabilize the backbone fold of the LB modules. In the modules of known structure, the isoleucine residu...

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