The Cell Line-Dependent Diversity in Initial Morphological Dynamics of Pancreatic Cancer Cell Peritoneal Metastasis Visualized by an Artificial Human Peritoneal Model.

BACKGROUND: Pancreatic ductal adenocarcinoma is considered as one of the most malignant types of cancer with rapid metastasis and invasion of the cancer cells, having peritoneal metastasis (PM) as a dominant factor of poor prognosis. Although the prevention of peritoneal dissemination would result in the inhibition of the initial metastatic process and contribute in improving the poor prognosis of the pancreatic cancer, the initial dynamics of PM are still unclear because of the lack of adequate models in studying the morphological and molecular details of pancreatic cancer cells. MATERIALS AND METHODS: The artificial human peritoneal tissue (AHPT) that can be applied in studying for the spatial dynamics of cancer PM in vitro has been established previously. In this study, the initial dynamics of the three pancreatic cell lines, undifferentiated carcinoma MIA PaCa-2, poorly differentiated adenocarcinoma Panc-1, and moderately differentiated adenocarcinoma BxPC3 on AHPT are examined. RESULTS: In a morphological analysis using light and electron microscopy, MIA PaCa-2 cells spread on the mesothelial layer with disruption of the sheet structure and infiltrated into the stroma-like tissue in AHPT. On the other hand, BxPC3 cells changed shapes from round into flat ones with rapid proliferation and formed sheet structure at the surface of the tissue replacing the mesothelial layer without vertical invasion into the tissue. Panc-1 cells demonstrated the intermediate characteristics of MIA PaCa-2 and BxPC3 on AHPT. These diverse morphological characteristics were verified by the correspondence with the results in a mouse model and were reflected by the profile of secreted oncogenic proteins of the three pancreatic cell lines. CONCLUSIONS: The initial dynamics in the peritoneal dissemination of these pancreatic cancer cell lines were demonstrated by AHPT, showing the morphological and molecular diversity depending on the degree of differentiation or the properties of oncogenic protein secretion.

Transplantation of artificial human lymphatic vascular tissues fabricated using a cell-accumulation technique and their engraftment in mouse tissue with vascular remodelling.

Transplantation of engineered tissues with microvascular structure is advancing towards therapeutic application to improve the flow of blood and/or lymphatic fluids. In lymphatic disorders, transplantation of tissue-engineered lymphatic grafts can be an ideal treatment for draining excessive lymphatic fluid. In this study, we examined the transplantation of 3-dimensional artificial human lymphatic network tissue (AHLT) fabricated by the cell accumulation technique into the subcutaneous tissue and fascia of mice. At 2 weeks after transplantation, the AHLT showed engraftment of artificial lymphatic vessels immunopositive for human CD31 and human podoplanin. Notably, we also observed the generation of blood vessel-like structure comprising endothelial cells immunopositive for human CD34 and mural-like cells immunopositive for human CD90 and αSMA, which were considered as myofibroblasts. In the fabrication of AHLT in vitro, the sporadic emergence of human CD34-positive/Prox-1-negative sites was observed, followed by the formation of blood vessel-like structure in the graft within 7 days after transplantation. The fine structure of engrafted AHLT observed by transmission electron microscopy showed that the engrafted artificial lymphatic vessels possess the specific structures of native lymphatic capillaries such as loose interendothelial connections and anchoring filaments. In contrast, blood vessel-like structure showed tight interendothelial connections, thick basement membranes, and layers of mural-like cells, which resemble small blood vessels. These results suggested the remodelling of artificial lymphatic network to form blood vessel-like structure associated with mural-like cells along with AHLT fabrication and engraftment.

Construction of artificial human peritoneal tissue by cell-accumulation technique and its application for visualizing morphological dynamics of cancer peritoneal metastasis.

Human peritoneum is composed of mesothelial monolayer and stromal tissue containing microvasculature. Dissemination and infiltration of cancer cells to the peritoneum result in cancer peritoneal metastasis which is an important prognostic factor of intraperitoneal or intrapelvic carcinoma. To elucidate an initial metastatic mechanism of cancer cells, in vitro human peritoneal models are demanded. In this study, we created a three-dimensional artificial human peritoneal tissue (AHPT) harboring the blood or lymphatic vascular network by cell-accumulation technique. Morphological analysis demonstrated that AHPT had mesothelial monolayer with polygonal flat cells with apical microvilli, and stroma-like structure containing fibroblasts surrounded by extracellular matrix and blood or lymphatic vascular network. To assess AHPT as a tool for cancer peritoneal metastasis model, colon and ovarian cancer cells (HT-29 and SKOV3) were seeded onto AHPT. HT-29 cells showed poor metastatic characteristics forming thick clusters in mesothelial layer without invasion into stroma-like structure. On the other hand, SKOV3 cells rapidly invaded intercellular spaces between mesothelial cells and then spread over the stroma-like structure accompanying lymphatic invasion, showing aggressive metastatic characteristics. These results demonstrated that the metastatic dynamics of cancer cells with different characteristics are able to visualized by AHPT, suggesting that this tissue can be a powerful tool for the basic research of cancer peritoneal dissemination and metastasis.

Transplantation of three-dimensional artificial human vascular tissues fabricated using an extracellular matrix nanofilm-based cell-accumulation technique.

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.

Construction of three-dimensional liver tissue models by cell accumulation technique and maintaining their metabolic functions for long-term culture without medium change.

Three-dimensional (3D) hepatocyte cultures have attracted much attention to obtain high biological functions of hepatocyte for pharmaceutical drug assessment. However, maintaining the high functions for over one month is still a key challenge although many approaches have been reported. In this study, we demonstrate for the first time simple and rapid construction of 3D-hepatocyte constructs by our cell accumulation technique and their high biological functions for one month, without any medium change. The human hepatocyte carcinoma (HepG2) cells were coated with ∼ 7 nm-sized extracellular matrix (ECM) films consisting of fibronectin (FN) and gelatin (G), and then incubated in cell culture insert to construct 3D-tissue constructs for 24 h. The thickness of obtained 3D-HepG2 constructs was easily controlled by altering seeding cell number and the maximum is over 100 µm. When a large volume of culture media was employed, the 3D-constructs showed higher mRNA expression of albumin and some cytochrome P450 (CYP) enzymes as compared to general two-dimensional (2D) culture. Surprisingly, their high cell viabilities (over 80%) and high mRNA expressions were successfully maintained without medium change for at least 27 days. These results demonstrate novel easy and rapid technique to construct 3D-human liver tissue models which can maintain their high functions and viability for 1 month without medium change.

Ultrastructure of blood and lymphatic vascular networks in three-dimensional cultured tissues fabricated by extracellular matrix nanofilm-based cell accumulation technique.

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.