Neural stem/progenitor cells (NSPCs) proliferate as aggregates in vitro, but the mechanism of aggregation is not fully understood. Here, we report that aggregation promotes the proliferation of NSPCs. We found that the proliferation rate was linear and depended on the size of the aggregate; that is, the population doubling time of the NSPCs gradually decreased as the diameter approached 250 lm and flattened to a nearly constant value beyond this diameter. Given this finding, and with the intent of enhancing the efficiency of human NSPC expansion, we induced the NSPCs to form aggregates close to 250 lm in diameter quickly by culturing them in plates with Ubottomed wells. The NSPCs formed aggregates effectively in the U-bottomed wells, with cell numbers approximately 1.5 times greater than those in the aggregates that formed spontaneously in flat-bottomed wells. In addition, this effect of aggregation involved cell-cell signaling molecules of the Notch1 pathway. In the U-bottomed wells, Hes1 and Hes5, which are target genes of the Notch signal, were expressed at higher levels than in the control, flat-bottomed wells. The amount of cleaved Notch1 was also higher in the cells cultured in the U-bottomed wells. The addition of g-secretase inhibitor, which blocks Notch signaling, suppressed cell proliferation in the U-bottomed wells. These results suggest that the three-dimensional architecture of NSPC aggregates would create a microenvironment that promotes the proliferation of human NSPCs. V V C 2006 Wiley-Liss, Inc.

To evaluate the degree of cellular dedifferentiation, subculture of chondrocytes was conducted on a surface coated with collagen type I at a density of 1.05 mg/cm(2). In the primary culture, most of the cells were round in shape on the collagen (CL) substrate, whereas fibroblastic and partially extended cells were dominant on the polystyrene plastic (PS) substrate. Stereoscopic observation revealed that the round-shaped cells on the CL substrate were hemispherical with nebulous and punctuated F-actin filaments, whereas the fibroblastic cells on the PS substrate were flattened with fully developed stress fibers. This suggested that cell polarization was suppressed during culture on the former substrate. Although serial passages of chondrocytes through subcultures on the CL and PS substrates caused a decrease in the number of round-shaped cells, the morphological change was appreciably suppressed on the CL substrate, as compared with that on the PS substrate. It was found that only round-shaped cells formed collagen type II, which supports the view that cellular dedifferentiation can be suppressed to some extent on the CL substrate. Three-dimensional cultures in collagen gel were performed with cells isolated freshly and passaged on the CL or PS substrate. Cell density at 21 days in the culture of cells passaged on the CL substrate was comparable to that in the culture of freshly isolated cells, in spite of a significant reduction in cell density observed in the culture of cells passaged on the PS substrate. In addition, histological analysis revealed that the expression of glycosaminoglycans and collagen type II was of significance in the collagen gel with cells passaged on the CL substrate, and likewise in the gel with freshly isolated cells. This indicated that the CL substrate could offer a monolayer culture system for expanding chondrocyte cells.

An intelligent culture system accompanied by automated operations (liquid transfer and cell passage) was newly developed to perform serial cultures of human skeletal muscle myoblasts. To realize a desired performance, a laminin-coated surface was applied to myoblast expansion in a culture flask. It was found that the laminin coating enhanced the overall growth ability attributable not to shortening of the doubling time but to prevention of differentiation toward myotube formation, compared with that on a conventional plain surface. In addition, the effects of seeding density and confluence degree on the growth were investigated quantitatively in terms of cell attachment and division as well as proliferative cell population in the culture on the laminin-coated surface. With increasing in seeding density, the number of proliferative cells decreased at the end of culture accompanied by an increase in the confluence degree, which caused poor attachment of the passaged cells on the surface in the subsequent culture. The quantitative analyses of these cell behaviors helped us determine the appropriate seeding density and attainable confluence degree during one passage, which were 1.0 x 10(3) cells/cm(2) and 0.5 as the initial and boundary conditions, respectively. An automated culture system that could manage two serial cultures by monitoring the confluence degree was constructed. The automated operation with the intelligent determination of the time for passage was successfully performed without serious loss of growth activity, compared with manual operation using conventional flasks. These results indicated that the monitoring of confluence degree is effective to perform the culture passage of myoblasts, being contributable to automating the cell expansion process.

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