Expression profiles of angiogenesis-related proteins in prevascular three-dimensional tissues using cell-sheet engineering
Introduction
Cell-sheet technology is a tissue engineering methodology without the use of biodegradable scaffolds [1], [2], [3], [4]. Cell-sheets are harvested from confluently cultured cells on temperature-responsive culture dishes covalently grafted with a temperature-responsive polymer, poly (N-isopropylacrylamide), by lowering culture temperature without by enzyme digestion [5], [6]. Additionally, due to the presence of intact deposited extracellular matrices that are produced during in vitro cultivation, harvested cell-sheets can be easily reattached to other surfaces such as other cell-sheets and the host tissues. Cell-sheet-based clinical therapies have been performed to the treatments for corneal epithelial disease [7], esophageal stricture after endoscopic submucosal dissection [8], and dilated cardiomyopathy [9]. Moreover, by using this technology, functional multi-layered three-dimensional (3-D) cell-dense tissues including cardiac, hepatic, and pancreatic tissues are also created by layering cell-sheets for regenerative medicine [10], [11], [12].
A rapid and sufficient vascularization is crucial for the survival and function of engineered 3-D cell-dense tissues after transplantation. The prefabrication of endothelial cell network assembly (ECNA) in engineered tissues before transplantation, which is known as in vitro prevascularization, has been attempted as an approach to achieving the vascularization [13]. ECs are three-dimensionally co-cultured with other cell types in vitro, resulted in the organization of these cells into ECNA that often contains lumens spontaneously. Moreover, these transplanted 3-D tissues can anastomose by developing interconnections to the blood vessels of host tissue [13], [14], [15]. Therefore, the in vitro prevascularization of engineered tissues is beneficial for inducing functional anastomosis with the host vasculature.
On the other hand, our laboratory has also reported that an original approach for initiating in vitro prevascularization in regenerative multi-layered 3-D tissues by a cell-sheet-based sandwich co-culture system using temperature-responsive culture dishes and a cell-sheet manipulator [16], [17]. Human umbilical vein endothelial cells (HUVECs) were sandwiched between cell-sheets such as myoblast sheets and fibroblast (FB) sheets, resulted in the creation of prevascular layered cell-sheets. In the case of prevascular quintuple-layered myoblast sheets, the networked HUVECs were connected to the host vessels at one week after transplantation, and the partially formed microvessels were found to contain erythrocytes in the engrafted cell-sheets. Therefore, our prevascularizing strategy contributes to the development of transplantable 3-D cell-dense tissues for regenerative medicine. However, the mechanism of neovascularization after the transplantation of prevascular layered cell-sheets is hardly understood.
This study created a scaffold-free prevascular 3-D tissue by sandwiching human aortic endothelial cells (HAECs) between human dermal FB sheets using a cell-sheet-based sandwich co-culture system and investigated the loci and progression of neovascularization after subcutaneous transplantation in immune-deficient rats. Furthermore, the secretion rates of angiogenesis-related proteins from ECNA-positive/-negative cell-sheets were investigated for understanding the mechanism of transplanted prevascular cell-sheet-induced neovascularization.
Section snippets
Cell culture
Human dermal fibroblasts (FBs) derived from neonatal foreskin and human aortic endothelial cells (HAECs) were purchased from Lonza (Walkersville, MD). Human FBs and HAECs were cultured at 37 °C in a humidified atmosphere of 95% air with 5% CO2 in a commercially-available growth media, FGM-2 (Lonza) and EGM-2 (Lonza), respectively.
Preparation of cell-sheet manipulator
A cell-sheet manipulator is composed of gelatin gel, a plunger devise, and a plunger-guiding cover (Fig. S1). The top surface of plunger is coated with hydrogel for
Creation of double-layered FB sheets having networked HAECs
A HAECs-sandwiching double-layered FB sheet prepared by a cell-sheet manipulator was reattached on an UpCell dish as illustrated in Figure S1. For investigating the behavior of HAECs inserted between two FB sheets, the localization of fluorescent-labeled HAECs was monitored throughout the culture period after preparation (Fig. 1A). Immediately after being sandwiched at day 0, inserted cells were observed sparsely in the double-layered FB sheet. One day after being sandwiched, the
Discussion
Neovascularization is crucial for the survival and function of engineered 3-D cell-dense tissues after transplantation. ECNA prefabrication in engineered tissues prior to transplantation is essential for a possible neovascularization in the early post-transplanted period. However, the neovascularization process via transplanted ECNA-positive tissues is known to be unclear. In this study, the loci and progression of neovascularization after transplantation were able to characterize by using
Conclusion
This study showed the loci and progression of neovascularization after subcutaneous transplantation of cell-sheet engineering-based prevascular 3-D tissues. Newly-formed microvascular plexus seemed to be guided by preformed ECNA in the transplanted cell-sheet was formed at nearby the host blood vessels within the first 3 days post-transplant. Moreover, this study demonstrated that ECNA-positive/-negative double-layered FB sheets were able to secrete angiogenesis-related proteins such as VEGF,
Acknowledgments
We are grateful to Dr. Norio Ueno in Tokyo Women's Medical University for English editing. The present work was partially supported by Grant-in-Aid for Scientific Research (C) (21500433), Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology “Cell Sheet Tissue Engineering Center (CSTEC)” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and the Japan Society for the Promotion
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