Reconstruction Of Tissue-engineered Renal Tissue In Vitro Using Collagen As Scaffold

Since kidney is one of the vital organs of human body, renal diseases have severe effect on patients'health and quality of life. Researchers explored many methods for the treatment of renal diseases. In them, dialysis and renal allotransplantation are most effective to replace kidney function of end-stage renal disease as well as the acute renal failure. However, dialysis replaces only a small fraction of normal kidney function.Whereas, renal allotransplantation is restrained by donor shortage, allograft failure, and long-term immunosuppression. Therefore, it is urgent to explore new methods to give a supplement to current methods. The emergence of tissue engineering brings new hopes for the functional and biological replacement of end-stage renal disease as well as acute renal failure. There are many tissues having been engineered until now.However, to tissue engineer a kidney in vitro is a formidable task because of its complex cell composition and structures. In rencent years, researchers made some comparatively large progresses, for example, the renal cells, which were isolated from early-stage cloned bovine fetus, were seeded on polymer scaffolds and then implanted into the nuclear donor animal. They could regenerate renal tissue. In another study, the implantation of kidney precursor cells derived from early embryo formed the functional renal structure. These in vivo results implicated that renal cells could reconstitute functional renal structures. But, there are no in vitro reconstruction of kidney consisting of both tubules and glomeruli applying the strategy of tissue engineering to date.In this paper, we established a scaffold system by using collagenâ… and Matrigel, and used MDCK cells as seeding cells to test the feasibility of this system in kidney tissue engineering. Then we used primary tubular epithelial cells as seeding cells to reconstruct tissue engineered renal tubule. Based on the two parts, we reconstructed tissue-engineered renal tissue by using neonatal rat mixed renal cells as seeding cells, collagen gel and Matrigel as scaffold and exerted static stretch to mimic strained force environment of normal organism. The histological observation, immunofluorescence and renal function of the constructs were detected aiming at checking the feasibility and stability of this engineering technique to construct renal tissue in vitro.The contents are three parts as follows:Part 1: Reconstruction of tissue-engineered renal tubules using MDCK cells as seeding cells.In this study, firstly, we used MDCK cells as seeding cells, typeâ… collagen gel supplanting with different concentration of Matrigel as scaffold to construct engineered renal tubule sheets. The growth and aggregation of MDCK cells as well as the development of tubules were observed under microscope. After 2 weeks'culture, the constructs were abserved by histological examination to determine the optical proportion of collagen and Matrigel. Results showed Matrigel played an important role in the developing of tubules, and tubular structures were formed only in the sheets supplanting with Matrigel, while the concentration of 10% was better for the motility rate of cells. Secondly, we exerted static stretch by a casting mold to test the effect of static stretch on the formation of tubules. During 2 weeks'culture time, the morphologic changes of the sheets and the histological differences were detected. Our results indicated the static stretch contributed to the cytoactivity and the formation of tubules as well as raising the quality of reconstructs.Part 2: Reconstruction of tissue-engineered renal tubules using primary neonatal rat renal epithelial cells.In this study, firstly, neonatal rat renal tubular epithelial cells were isolated by sieving and enzyme-digesting methods. Immunochemistry was used to identify the epithelial cells. Secondly, we reconstructed the engineered renal tubular sheet using neonatal renal epithelial cells as seeding cells, typeâ… collagen gel supplanting with 10% Matrigel as scaffold and exerted static stretch on the sheets during culture. The histology, immunofluorescence, and ultrastructure were used to analyse the formation of tubules in the reconstructed sheets at the time points of 1 week and 2 weeks. The results showed that during in vitro culture, the constructs shrinked and formed a butterfly-like shape. Cells in the sheets expressed epithelial cell-surface marker of CK18. They acquired polarity, and formed three dimensional tubules.Part 3: Reconstruction of tissue-engineered renal tissue using primary mixed neonatal rat renal cells.In this study, we reconstructed renal tissue using mixed renal cells as seeding cells, typeâ… collagen gel supplanting with 10% Matrigel as scaffold and exerted static stretch on the sheets. The histology, immunofluorescence, ultrastructure and renal functin of the sheets were analysed at the time points of 1 week, 2 weeks and 3 weeks. The results demonstrated neonatal rat renal cells survived well in the gel, and self-assembled to form tubules and the glomeruli-like structures. The concentration of urea nitrogen and creatinine altered during culture, which might mean that the newly formed renal structures had certain functions.In conclusion, we reconstructed tissue-engineered renal tissue by using MDCK cell and neonatal rat renal epithelial cells as well as mixed neonatal renal cells as seeding cells respectively, collagenâ… supplanting with Matrigel as scaffold and exerted static stretch on the sheets during culture. The results demonstrated that renal cells self-assembled to form tubules and glomeruli-like structures in tissue engineered renal sheet. The tissue-engineered renal tissue could be used as models to investigate the development of kidney and to screen drugs for kidney diseases. With further investigation and optimization, they also had potential perspective in clinical application. More important, this study provided a strategy for the reconstruction of other vital organs.