Tumour models mimicking in vivo three-dimensional (3D) microenvironments are of increasing interest in drug discovery because of the limitations inherent to current models. For example, preclinical assays that rely on monolayer or spheroid cell cultures cannot easily predict 3D cancer behaviours because they have no vasculature. Furthermore, there are major differences in cancer behaviour between human and animal experiments. Here, we show the construction of 3D blood/lymph-vascularized human stromal tissues that can be combined with cancer cells to mimic dynamic metastasis for real-time throughput screening of secreted proteinases. We validated this tool using three human carcinoma cell types that are known to invade blood/lymph vessels and promote angiogenesis. These cell types exhibited characteristic haematogenous/lymphogenous metastasis and tumour angiogenesis properties. Importantly, these carcinoma cells selectively secreted different matrix metalloproteinases depending on their metastasis stage and target vasculature, suggesting the possibility of developing drugs that can target each secreted proteinase. We conclude that the 3D tissue tool will be a powerful throughput system for predicting cancer cell responses and time-dependent secretion of molecules in preclinical assays.

Cell accumulation technique is an extracellular matrix (ECM) nanofilm-based tissue-constructing method that enables formation of multilayered hybrid culture tissues. In this method, ECM-nanofilm is constructed using layer-by-layer assembly of fibronectin and gelatin on culture cells. The ECM-nanofilm promotes cell-to-cell interaction; then the three-dimensional (3D) multilayered tissue can be constructed with morphological change of the cells mimicking living tissues. By using this method, we have successfully produced tubular networks of human umbilical venous endothelial cells (HUVECs) and human dermal lymphatic endothelial cells (HDLECs) in 3D multilayered normal human dermal fibroblasts (NHDFs). This study demonstrated morphological characteristics of the vascular networks in the engineered tissues by using light and electron microscopy. In light microscopy, HUVECs and HDLECs formed luminal structures such as native blood and lymphatic capillaries, respectively. Electron microscopy showed distinct ultrastructural aspects of the vasculature of HUVECs or HDLECs with intermediated NHDFs and abundant ECM. The vasculature constructed by HUVECs exhibited structures similar to native blood capillaries, involving overlapping endothelial connections with adherens junctions, abundant vesicles in the endothelial cells and basement membrane-like structure. The detection of laminin around HUVEC-constructed vessels supported the presence of perivascular basal lamina. The vasculature constructed by HDLECs showed some ultrastructural characteristics similar to those of native lymphatic capillaries such as irregular vascular shape, loose adhesive connection and gap formation between endothelial cells. In conclusion, our novel vascular network models fabricated by the cell accumulation technique provide highly organized blood and lymphatic capillary networks mimicking the vasculatures in vivo.

We have established a novel three-dimensional (3D) tissue-constructing technique, referred to as the 'cell-accumulation method', which is based on the self-assembly of cultured human cells. In this technique, cells are coated with fibronectin and gelatin to construct extracellular matrix (ECM) nanofilms and cultured to form multi-layers in vitro. By using this method, we have successfully fabricated artificial tissues with vascular networks constructed by co-cultivation of human umbilical vein-derived vascular endothelial cells between multi-layers of normal human dermal fibroblasts. In this study, to assess these engineered vascular tissues as therapeutic implants, we transplanted the 3D human tissues with microvascular networks, fabricated based on the cell-accumulation method, onto the back skin of nude mice. After the transplantation, we found vascular networks with perfusion of blood in the transplanted graft. At the boundary between host and implanted tissue, connectivity between murine and human vessels was found. Transmission electron microscopy of the implanted artificial vascular tubules demonstrated the ultrastructural features of blood capillaries. Moreover, maturation of the vascular tissues after transplantation was shown by the presence of pericyte-like cells and abundant collagen fibrils in the ECM surrounding the vasculature. These results demonstrated that artificial human vascular tissues constructed by our method were engrafted and matured in animal skin. In addition, the implanted artificial human vascular networks were connected with the host circulatory system by anastomosis. This method is an attractive technique for engineering prevascularized artificial tissues for transplantation. Copyright © 2015 John Wiley & Sons, Ltd.

Some free-living flatworms in the phylum Platyhelminthes possess strong regenerative capability that depends on putative pluripotent stem cells known as neoblasts. These neoblasts are defined based on several criteria, including their proliferative capacity and the presence of cellular components known as chromatoid bodies. Polyclads, which are marine flatworms, have the potential to be a good model system for stem cell research, yet little information is available regarding neoblasts and regeneration. In this study, transmission electron microscopy and immunostaining analyses, using antibodies against phospho-histone H3 and BrdU, were used to identify two populations of neoblasts in the polyclad Notoplana humilis: mesodermal neoblasts (located in the mesenchymal space) and gastrodermal neoblasts (located within the intestine, where granular club cells and phagocytic cells are also located). Light and electron microscopic analyses also suggested that phagocytic cells and mesodermal/gastrodermal neoblasts, but not granular club cells, migrated into blastemas and remodeled the intestine during regeneration. Therefore, we suggest that, in polyclads, intestinal regeneration is accomplished by mechanisms underlying both morphallaxis (remodeling of pre-existing tissues) and epimorphosis (de novo tissue formation derived from mesodermal/gastrodermal neoblasts). Based on the assumption that gastrodermal neoblasts, which are derived from mesodermal neoblasts, are intestinal stem cells, we propose a model to study intestinal regeneration.