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1.
Extracellular matrix (ECM) has been focused to understand tumor progression in addition to the genetic mutation of cancer cells. Here, we prepared “staged tumorigenesis-mimicking matrices” which mimic in vivo ECM in tumor tissue at each malignant stage to understand the roles of ECM in tumor progression. Breast tumor cells, MDA-MB-231 (invasive), MCF-7 (non-invasive), and MCF-10A (benign) cells, were cultured to form their own ECM beneath the cells and formed ECM was prepared as staged tumorigenesis-mimicking matrices by decellularization treatment. Cells showed weak attachment on the matrices derived from MDA-MB-231 cancer cells. The proliferations of MDA-MB-231 and MCF-7 was promoted on the matrices derived from MDA-MB-231 cancer cells whereas MCF-10A cell proliferation was not promoted. MCF-10A cell proliferation was promoted on the matrices derived from MCF-10A cells. Chemoresistance of MDA-MB-231 cells against 5-fluorouracil increased on only matrices derived from MDA-MB-231 cells. Our results showed that the cells showed different behaviors on staged tumorigenesis-mimicking matrices according to the malignancy of cell sources for ECM preparation. Therefore, staged tumorigenesis-mimicking matrices might be a useful in vitro ECM models to investigate the roles of ECM in tumor progression.  相似文献   
2.
Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.  相似文献   
3.
目的:阐明小鼠去细胞拟胚体对小鼠Lewis肺癌细胞在体内生长的影响。方法:先制备来源于小鼠胚胎干细胞的拟胚体,然后用SDS去细胞处理。实验分成3组:小鼠Lewis肺癌细胞与去细胞拟胚体培养组,癌细胞与Matrigel培养组和单纯癌细胞组(每组n=12)。培养3天后注射入裸鼠体内,观察肿瘤生长情况。28天取出瘤体,Ki67和CD31免疫组化染色(n=12)检测细胞增殖和肿瘤微血管密度(MVD),Western blot检测组织Paxillin,E-cadherin和β-actin水平(n=6)。结果:去细胞拟胚体组肿瘤生长明显较单纯细胞组和Matrigel组慢。去细胞拟胚体组Ki67指数((17.1±2.6)%)明显小于单纯细胞组((34.5±4.7)%)和Matrigel组((48.4±8.6)%)(P0.05);去细胞拟胚体组的MVD(18.7±3.6个/mm2)明显小于单纯细胞组(32.1±6.4个/mm2)和Matrigel组(42.6±7.1个/mm2)(P0.05)。Western blot结果提示去细胞拟胚体组的Paxillin表达小于单纯细胞组和Matrigel组(P0.05),而E-cadherin表达大于单纯细胞组和Matrigel组(P0.05)。结论:小鼠去细胞拟胚体对小鼠Lewis肺癌细胞在体内有明显的促分化作用。  相似文献   
4.
Xenogeneic tissues are derived from other animal species and provide a source of material for engineering mechanically functional tissue grafts, such as heart valves, tendons, ligaments, and cartilage. Xenogeneic tissues, however, contain molecules, known as antigens, which invoke an immune reaction following implantation into a patient. Therefore, it is necessary to remove the antigens from a xenogeneic tissue to prevent immune rejection of the graft. Antigen removal can be accomplished by treating a tissue with solutions and/or physical processes that disrupt cells and solubilize, degrade, or mask antigens. However, processes used for cell and antigen removal from tissues often have deleterious effects on the extracellular matrix (ECM) of the tissue, rendering the tissue unsuitable for implantation due to poor mechanical properties. Thus, the goal of an antigen removal process should be to reduce the antigen content of a xenogeneic tissue while preserving its mechanical functionality. To expand the clinical use of antigen-removed xenogeneic tissues as biomechanically functional grafts, it is essential that researchers examine tissue antigen content, ECM composition and architecture, and mechanical properties as new antigen removal processes are developed.  相似文献   
5.
侯楠  朱力 《生物磁学》2011,(2):381-383
去细胞基质在组织工程及再生医学的大量应用为解决组织器官的修复和重建等难题带来了希望。去细胞方法大致可以分为三类:化学处理法、物理处理法及酶学处理法,且已经应用于组织工程及再生医学的各个方面。本文总结并分类目前常用的去细胞方法及其在组织工程各方面的应用,对目前国内外常用的去细胞方法及其在组织工程及再生医学中的应用进行回顾总结与分析。  相似文献   
6.
Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.  相似文献   
7.
The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.  相似文献   
8.

Background

Numerous proteins and small leucine-rich proteoglycans (SLRPs) make up the composition of the extracellular matrix (ECM). Assembly of individual fibrillar components in the ECM, such as collagen, elastin, and fibronectin, is understood at the molecular level. In contrast, the incorporation of non-fibrillar components and their functions in the ECM are not fully understood.

Scope of review

This review will focus on the role of the matricellular protein thrombospondin (TSP) 2 in ECM assembly. Based on findings in TSP2-null mice and in vitro studies, we describe the participation of TSP2 in ECM assembly, cell–ECM interactions, and modulation of the levels of matrix metalloproteinases (MMPs).

Major conclusions

Evidence summarized in this review suggests that TSP2 can influence collagen fibrillogenesis without being an integral component of fibrils. Altered ECM assembly and excessive breakdown of ECM can have both positive and negative consequences including increased angiogenesis during tissue repair and compromised cardiac tissue integrity, respectively.

General significance

Proper ECM assembly is critical for maintaining cell functions and providing structural support. Lack of TSP2 is associated with increased angiogenesis, in part, due to altered endothelial cell–ECM interactions. Therefore, minor changes in ECM composition can have profound effects on cell and tissue function. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.  相似文献   
9.
Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.  相似文献   
10.
The multidisciplinary research of tissue engineering utilizes biodegradable or decellularized scaffolds with autologous cell seeding. Objective of this study was to investigate the impact of different decellularization protocols on extracellular matrix integrity of xenogeneic tissue by means of multiphoton femtosecond laser scanning microscopy, biochemical and histological analysis. Pulmonary valves were dissected from porcine hearts and placed in a solution of trypsin-EDTA and incubated at 37 degrees C for either 5, 8, or 24 h, followed by a 24 h PBS washing. Native and decellularized valves were processed for histology, DNA, cell proliferation, matrix proteins and biomechanical testing. Multiphoton NIR laser microscopy has been applied for high-resolution 3D imaging of collagen and elastin. Distinct differences in several ECM components following decellularization time were observed. Total GAG contents decreased in a time-dependent manner, with o-sulfated GAGs being more susceptible to degradation than n-sulfated GAGs. Efficiency of insoluble collagen extraction increased proportionally with decellularization time, suggesting ECM-integrity may be compromised with prolonged incubation. Biomechanical testing revealed a gradual weakening of mechanical strength with increased decellularization time. The enzymatic decellularization process of heart valves revealed a time-dependent loss of cells, ECM components and biomechanical strength. In order to avoid any immune response a thorough decellularization of 24 h remains mandatory.  相似文献   
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