High-density, three-dimensional cell cultures are difficult to grow in vitro. The rotating-wall vessel (RWV) described here has cultured BHK-21 cells to a density of 1.1 X 10(7) cells/ml. Cells on microcarriers were observed to grow with enhanced bridging in this batch culture system. The RWV is a horizontally rotated tissue culture vessel with silicon membrane oxygenation. This design results in a low-turbulence, low-shear cell culture environment with abundant oxygenation. The RWV has the potential to culture a wide variety of normal and neoplastic cells.

BHK-21 cells were cultured under various shear stress conditions in an Integrated Rotating-Wall Vessel (IRWV). Shear ranged from 0.5 dyn/cm2 (simulated microgravity) to 0.92 dyn/cm2. Under simulated microgravity conditions, BHK-21 cells complexed into three-dimensional cellular aggregates attaining 6 x 10(6) cells/ml as compared to growth under 0.92 dyn/cm2 conditions. Glucose utilization in simulated microgravity was reduced significantly, and cellular damage at the microcarrier surface was kept to a minimum. Thus, the integrated rotating wall vessel provides a quiescent environment for the culture of mammalian cells.

Growth patterns of a number of human tumor cell lines that from three-dimensional structures of various architectures when cultured without carrier beads in a NASA rotary cell culture system are described and illustrated. The culture system, which was designed to mimic microgravity, maintained cells in suspension under very low-shear stress throughout culture. Spheroid (particulate) production occurred within a few hours after culture was started, and spheroids increased in size by cell division and fusion of small spheroids, usually stabilizing at a spheroid diameter of about 0.5 mm. Architecture of spheroids varied with cell type. Cellular interactions that occurred in spheroids resulted in conformation and shape changes of cells, and some cell lines produced complex, epithelial-like architectures. Expression of the cell adhesion molecules, CD44 and E cadherin, was upregulated in the three-dimensional constructs. Coculture of fibroblast spheroids with PC3 prostate cancer cells induced tenascin expression by the fibroblasts underlying the adherent prostate epithelial cells. Invasion of the fibroblast spheroids by the malignant epithelium was also demonstrated.

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3- While important advances have been made toward understanding how Salmonella interacts with the intestinal epithelium to initiate disease (reviewed in references 6 and 44), investigations into the interaction of Salmonella with the human intestinal epithelium have been limited by the lack of in vitro and in vivo models which faithfully replicate the in vivo condition. In particular, it is well documented that important differences exist between the pathogenesis of Salmonella enterica serovar Typhimurium in human infections and that in widely used cell culture and animal models (34,40,47). In vitro assays using cultured mammalian epithelial cells have long been used as a model for investigating the interaction between Salmonella and the intestinal mucosa. However, there are inherent limitations associated with the use of these cultured cell lines (34), as they are not exact models of the conditions faced in vivo by Salmonella. Several characteristics of conventional tissue culture models have raised concerns regarding their overall efficacy as models for microbial infectivity in general (34) due to the dedifferentiation of these cells during conventional cell culture. Indeed, many of the physiological differences between cultured cells and their in vivo counterparts are believed to be the result of the dissociation of cells from their native three-dimensional (3-D) geometry in vivo to their propagation on a two-dimensional substrate in vitro (10). Likewise, many characteristics of animal models fail to mimic the human disease, and animal models present a complex system in which many variables cannot be controlled. A high-fidelity enteric cell culture model could provide new insights into studies of Salmonella infectivity by bridging the gap between the inherent limitations of cultured mammalian cells and intact animals.DFor humans, S. enterica serovar Typhimurium is among the most common Salmonella serotypes isolated from individuals suffering from infectious gastroenteritis and has long been recognized as a major public health problem (23). Gastroenteritis results from infection of the small intestine after ingestion with Salmonella. Indeed, the ability to colonize the intestinal epithelium is an essential feature in the pathogenicity of Salmonella infection. Moreover, the initial interactions between Salmonella and the host intestinal epithelium are believed to play a key role in mediating the intense inflammatory and secretory response which is a hallmark of serovar Typhimurium infections in humans (reviewed in reference 6). Studies with cultured intestinal epithelial cells have shown that,

No abstract