We investigated the interactions between HeLa cells and human umbilical vein endothelial cells (HUVECs) by monitoring their movements in a controllable coculture system. Two complementary, detachable, cell-substrates, one of polystyrene (PS) and the other of poly(dimethylsiloxane) (PDMS), were fabricated by replica molding. Coculturing was started by mechanically assembling two complementary substrates. One substrate was covered with a confluent layer of HeLa cells and its complement covered with confluent HUVECs. Using this coculture system as a tumor/endothelium model, we found that the HeLa cells migrated towards the HUVECs, while, simultaneously, the HUVECs retreated and that both types of cells migrated approximately twice as rapidly (two hundred microns per twenty-four hours) as they did alone. Additionally, when direct contact between the two cell types was prevented, the HUVECs initially migrated towards the HeLa cells and then retreated. The characteristics of the cell movements, i.e. direction and speed, probably are consequences of cell-cell signaling, with such signals possibly important during tumor cell intra- and extravasation.

A microfluidic device was integrated with a controlled coculture system of HeLa cells and human umbilical vein endothelial cells (HUVECs). This integrated assembly allowed control of the direction of flow of medium (along with signaling factors secreted from cells) across the cultured cells. We grew HeLa cells and HUVECs to confluency on separate substrates and then joined the two substrates. A microfluidic device was then assembled onto the substrates and a cell coculture was initiated with controlled perfusion of the medium. When the medium flow was directed from the HeLa side to the HUVEC side, the HUVECs retreated and the HeLa cells migrated into the newly vacated areas. By contrast, when the medium flow was in the opposite direction, there was essentially no net movement of either cell type. Our results suggest that the migration of HeLa cells and HUVECs in coculture was likely mediated by soluble factors produced by HeLa cells.

Abstract. The cabbage armyworm, Mamestra brassicae, enters diapause in the early pupal stage. Pupal diapause is induced by rearing the larvae under short day lengths. We previously demonstrated that feeding Dopa during last larval instar induces pupal dia pause even under long day lengths. In order to elucidate the mechanism by which pupal diapause is induced after experiencing short day lengths or fed Dopa under long day lengths, we analyzed gene expression in the brain of M. brassicae larvae under both of these conditions using a subtractive hybridization technique. After the secondary screen, 49 clones and 28 clones were identified as short day length or Dopa-feeding specific clones, respectively. All of these genes were sequenced and, using the base sequences of these clones, primers were synthesized. To confirm the genes enhanced specifically by these conditions, quantitative real-time PCR was carried out. This quantitative PCR analysis identified 15 and 1 clone whose expression was enhanced by the short day length condi tions or Dopa-feeding, respectively. Among these clones, the gene with a high level of identity to receptor for activated protein kinase C (RACK) from Heliothis virescens is the most dramatically up-regulated under both conditions.

Two-dimensional cell patterns prepared on substrate surfaces by an electrochemical-based biolithography method have been transferred into fibrin gels prepared in situ. Line patterns of human umbilical vein endothelial cells (HUVECs) in the gel that was strained after the transfer formed a linear vessel-like structure within 8 days.

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