Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after approximately 10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (+/-0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (+/-0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.

Endothelial progenitor cell (EPC) transplantation is a promising treatment option for ischemic diseases, and understanding the mechanism mediating angiogenic effects of EPCs is essential to further improve its effectiveness as well as their application to in vitro tissue engineering. Here we investigated EPC contribution on network formation using an in vitro three-dimensional network model. Bovine microvascular endothelial cells (ECs) were seeded on collagen gel to create a three-dimensional network model. Subsequently, EPCs isolated from rat bone marrow were seeded on top of a confluent endothelial cell (EC) monolayer to create EPC+EC model. In this model, EPCs promoted EC network formation. Quantitative analyses revealed that the total length, number, and depth of networks were significantly enhanced with the addition of EPCs. EPCs tended to localize in networks formed inside collagen gel rather than on a confluent monolayer with increasing experimental duration. In addition, EPCs preferentially distributed near sprout positions in a confluent monolayer. Furthermore, EPC-conditioned medium promoted network formation, and vascular endothelial growth factor was detected in the conditioned medium. Taken together, EPCs contributed to network formation by direct incorporation with on-site growth factor secretion. The angiogenic ability of EPCs offers a possible cell source to reconstruct vascularized tissues in vitro.

Neuroblastoma, which is a malignant tumor consisting of dedifferentiated neuroectodermal cells, is known to show spontaneous maturation or regression in its growth. Cultured human neuroblastoma GOTO cells could be induced to differentiate into Schwannian cells and neuronal cells by incubation in the presence of 5-bromo-2'-deoxyuridine (BrdU) and by serum depletion, respectively. Here we report that in association with these differentiations, cells differentiated into Schwannian cells specifically expressed a cell adhesion molecule CD44, of which expression is usually suppressed in GOTO cells. In contrast, it remained suppressed in cells differentiated into neuronal cells. Polymerase-chain reaction revealed that the CD44 species expressed was the hemopoietic form (CD44H) with long cytoplasmic tail. Furthermore, the newly expressed CD44 in the cells was found exclusively in membrane microdomains, called lipid rafts. These data suggest that CD44 might play an important role in GOTO cells differentiated into Schwannian cells.

We have developed a novel method for screening hybridoma clones producing antibodies against plasma membrane-associated materials. The method is based on the binding of cells by antigen-antibody complex formation to immobilized antibodies. The monoclonal antibodies (MAbs) were trapped by goat anti-mouse immunoglobulin antibodies, which were coated on the surface of 96-well plastic plates. Cells to be tested were then added to these plates and the number of bound cells was quantified by the activity of phosphatase of bound cells. This method allows quick and reliable screening of hybridoma clones without resorting to the use of expensive instruments such as a flow cytometer. We immunized mice with two different kinds of cells and obtained two interesting but contrasting MAbs reacting to cell-surface antigens: one interacts quite specifically with neuroblastoma cells and the other reacts equally with various cells with the exception of GOTO, a neuroblastoma cell.