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Methods in Molecular Biology

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https://www.readbyqxmd.com/read/28921421/tissue-engineering-of-3d-organotypic-microtissues-by-acoustic-assembly
#1
Yuqing Zhu, Vahid Serpooshan, Sean Wu, Utkan Demirci, Pu Chen, Sinan Güven
There is a rapidly growing interest in generation of 3D organotypic microtissues with human physiologically relevant structure, function, and cell population in a wide range of applications including drug screening, in vitro physiological/pathological models, and regenerative medicine. Here, we provide a detailed procedure to generate structurally defined 3D organotypic microtissues from cells or cell spheroids using acoustic waves as a biocompatible and scaffold-free tissue engineering tool.
September 19, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28889353/the-detection-techniques-for-autophagy-associated-cell-death-related-genes-and-proteins-gene-expression-assay-and-immunohistochemistry
#2
Melek Ozturk, Ozlem Dagdeviren Ozsoylemez, Fatma Kaya Dagistanli
Autophagy is important in cellular homeostasis for the cell survival mechanism. Deficiency or excess of autophagy is generally related to some of diseases such as cancer and neurodegeneration. Although autophagy is a cell survival mechanism, it can mediate programmed cell death in several conditions. Autophagy-related genes (ATGs) regulate the autophagy and also control the crosstalk with autophagy-associated cell death and apoptosis in some condition. Various methods have been used to detect the marker genes and the proteins involved in these processes...
September 10, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28856614/decellularized-liver-scaffold-for-liver-regeneration
#3
Wei Yang, Renpei Xia, Yujun Zhang, Hongyu Zhang, Lianhua Bai
After being initially hailed as the ultimate solution to end-stage organ failure, such as end-stage liver disease (ESLD), engineering of vascularized tissues has stalled because of the need for a well-structured circulatory system that can maintain the cells to be seeded inside the construct.In the field of regenerative medicine, decellularized scaffolds, derived mainly from various non-autologous whole organs, have become an emerging treatment technique to overcome this obstacle. As a result of significant progress made in recent years, organogenesis through whole-organ decellularization scaffolds may now become more feasible than ever before...
August 31, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28842896/determination-of-autophagy-in-the-caco-2-spontaneously-differentiating-model-of-intestinal-epithelial-cells
#4
Sinem Tunçer, Sreeparna Banerjee
The Caco-2 colorectal cancer cell line is widely used as a model for intestinal differentiation and barrier function. These cells, upon reaching confluency, spontaneously differentiate into enterocyte-like cells, synthesize intestinal enzymes, and form domes. Caco-2 cells also undergo autophagy in the course of differentiation. The criteria to establish the induction of autophagy in cells are already well established. Here, we describe the protocol for the spontaneous differentiation of Caco-2 cells and the detection of autophagy using Western blot, flow cytometry, and immunofluorescence...
August 27, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28842895/skeletal-muscle-lysosomal-function-via-cathepsin-activity-measurement
#5
Kristyn Gumpper, Matthew Sermersheim, Michael X Zhu, Pei-Hui Lin
Muscle wasting or cachexia is commonly associated with aging and many diseases such as cancer, infection, autoimmune disorders, and trauma. Decrease in muscle mass, or muscle atrophy, is often caused by dysfunction of protein proteolytic systems, such as lysosomes, which regulate protein turnover and homeostasis. Lysosomes contain many hydrolases and proteases and, thus, represent the major organelle that control protein turnover. Recently, lysosomes have emerged as a signaling hub to integrate cellular functions of nutrient sensing and metabolism, autophagy, phagocytosis, and endocytosis, which are all related to tissue homeostasis...
August 27, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28815517/autophagy-in-adipocyte-differentiation
#6
Zhipeng Tao, Longhua Liu, Louise D Zheng, Zhiyong Cheng
Adipose tissue regulates metabolic homeostasis by acting as an endocrine organ and energy reservoir. Adipose tissue development and functional maintenance are dependent on adipocyte differentiation, in which autophagy plays an important role. It has been shown that autophagy deficiency dampens adipocyte differentiation, compromises adipose tissue development, dysregulates adipocytokine secretion, and even causes sudden death in young animals. Therefore, accurate assessment of autophagy in adipocyte is critical for the study of adipose biology or pathology of metabolic diseases...
August 17, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28815516/gastrointestinal-epithelial-organoid-cultures-from-postsurgical-tissues
#7
Soojung Hahn, Jongman Yoo
An organoid is a cellular structure three-dimensionally (3D) cultured from self-organizing stem cells in vitro, which has a cell population, architectures, and organ specific functions like the originating organs. Recent advances in the 3D culture of isolated intestinal crypts or gastric glands have enabled the generation of human gastrointestinal epithelial organoids. Gastrointestinal organoids recapitulate the human in vivo physiology because of all the intestinal epithelial cell types that differentiated and proliferated from tissue resident stem cells...
August 17, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28808979/decellularization-of-liver-and-organogenesis-in-rats
#8
Nana Shirakigawa, Hiroyuki Ijima
Recently, organ construction has been attempted using decellularized organs. In this study, we used decellularized rat liver to construct liver tissue by recellularization. The right lobe of the rat liver was decellularized with 4% Triton X-100 solution, recellularized with 10(7) rat hepatocytes, and albumin synthesis in the recellularized right lobe was observed. Therefore, we introduce a method of decellularizing rat liver, which retains its fine vascular structure after removal of all the cells, perform organogenesis using the decellularized liver, and evaluate the structural and functional properties of the products...
August 15, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28798993/decellularization-and-recellularization-of-cartilage
#9
Catherine A Bautista, Bahar Bilgen
Decellularization of cartilage enables the use of cartilage allografts or xenografts as natural scaffolds for repair and regeneration of injured cartilage. The preservation of the extracellular matrix ultrastructure of the graft makes this a promising tool for cartilage tissue engineering. We have optimized the decellularization protocol by enzymatically digesting proteoglycans while preserving the native collagen architecture. Here we describe our methods for cartilage decellularization and cell labeling for the tracking of infiltration for recellularization in detail...
August 11, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28795365/preparation-of-cell-derived-decellularized-matrices-mimicking-native-ecm-during-the-osteogenesis-and-adipogenesis-of-mesenchymal-stem-cells
#10
Takashi Hoshiba, Naoki Kawazoe, Guoping Chen
The extracellular matrix (ECM) is an important extracellular microenvironmental factor that regulates stem cell differentiation. The ECM is remodeled according to stem cell differentiation progression to precisely regulate the differentiation. Thus, it is expected that the matrices mimicking native ECM surrounding differentiating cells at each differentiation stage provide a favorable microenvironment to promote stem cell differentiation. However, it is difficult to prepare matrices mimicking native ECM using chemical methods because the ECM has a complicated composition...
August 10, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28786033/wharton-s-jelly-matrix-decellularization-for-tissue-engineering-applications
#11
Gabriel L Converse, Dandan Li, Eric E Buse, Richard A Hopkins, Omar S Aljitawi
Scaffolds, both natural and synthetic, used in tissue engineering provide mechanical support to cells. Tissue decellularization has been used to provide natural extracellular matrix scaffolds for tissue engineering purposes. In this chapter we focus on describing the methodology used to decellularize Wharton's jelly matrix, the mucous connective tissue that surrounds umbilical cord vessels, to obtain decellularized Wharton's jelly matrix (DWJM); an extracellular matrix that can be used for tissue engineering purposes...
August 8, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28776178/protocols-for-rat-uterus-isolation-and-decellularization-applications-for-uterus-tissue-engineering-and-3d-cell-culturing
#12
Arvind Manikantan Padma, Tom Tristan Tiemann, Ahmed Baker Alshaikh, Randa Akouri, Min Jong Song, Mats Hellström
Sophisticated culturing conditions are required to grow cells in a three-dimensional (3D) environment. Cells then require a type of scaffold rich in proteins, growth factors, and signaling molecules that simulates their natural environment. Tissues from all species of animals have an organ-specific extracellular matrix (ECM) structure that plays a key role in cell proliferation and migration. Hence, the scaffold composition plays a significant role for any successful 3D cell culturing system. We developed a whole rat uterus ECM scaffold by the perfusion of detergents and ionic solutions through the vascular system of an isolated normal rat uterus in a process termed "decellularization...
August 4, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28770494/cell-microencapsulation-in-polyethylene-glycol-hydrogel-microspheres-using-electrohydrodynamic-spraying
#13
Mozhdeh Imaninezhad, Era Jain, Silviya Petrova Zustiak
Microencapsulation of cells is beneficial for various biomedical applications, such as tissue regeneration and cell delivery. While a variety of techniques can be used to produce microspheres, electrohydrodynamic spraying (EHS) has shown promising results for the fabrication of cell-laden hydrogel microspheres in a wide range of sizes and in a relatively high-throughput manner. Here we describe an EHS technique for the fabrication of cell-laden polyethylene glycol (PEG) microspheres. We utilize mild hydrogel gelation chemistry and a combination of EHS parameters to allow for cell microencapsulation with high efficiency and viability...
August 3, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28770493/measurement-of-lysophosphatidic-acid-and-sphingosine-1-phosphate-by-liquid-chromatography-coupled-electrospray-ionization-tandem-mass-spectrometry
#14
Maria P Kraemer, Suchismita Halder, Susan S Smyth, Andrew J Morris
Lysophosphatidic acids and sphingosine-1-phosphate are bioactive lipids that regulate diverse cellular and physiological processes through actions that are largely mediated by cell surface receptors. The roles played by these lipids in multiple disease processes make the enzymes and receptors involved in their synthesis, inactivation, and signaling attractive targets for pharmacological therapies. In this chapter we describe methods for sensitive accurate quantitation of LPA and S1P levels in biological fluids using liquid chromatography-coupled electrospray ionization tandem mass spectrometry...
August 3, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28770492/decellularized-bone-matrix-scaffold-for-bone-regeneration
#15
Guobao Chen, Yonggang Lv
Decellularized bone matrix is gaining a lot of attention as implantable biomaterials and/or biological scaffolds for bone tissue repair, and shows good clinical performance. This chapter describes the processing techniques and characterization protocols of decellularized bone. For the applications of the decellularized bone scaffold in promoting bone repair and regeneration, we discuss some of the current advances, and highlight the advantages and disadvantages of these scaffolds. Fabrication and application of the hydrogel derived from decellularized bone for bone tissue engineering are also presented...
August 3, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28770491/gene-expression-array-analysis-to-identify-candidate-tumor-suppressor-genes-in-melanoma
#16
Mitchell S Stark, Vanessa F Bonazzi
Melanoma is a complex multifactorial disease; therefore, a combination of various approaches is necessary to girt all aspects of its biology and identify the many different genes and factors involved in its etiology.Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. Here, we describe a new pipeline based on an integrative and comparative analysis of several array platform, post-demethylation treatment expression data, methylation array, and constitutive mRNA expression analysis to identify novel TSGs frequently methylated in melanoma...
August 3, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28755279/in-vitro-methods-to-study-the-modulation-of-migration-and-invasion-by-sphingosine-1-phosphate
#17
Melina G Castro, Ludmila E Campos, Yamila I Rodriguez, Sergio E Alvarez
Sphingosine-1-phosphate (S1P) is a bioactive lipid that modulates migratory behavior of cells during embryonic development. In addition, S1P might promote tumor progression by enhancing migratory ability and invasiveness of tumor cells. Migration is a complex process that implies cytoskeletal reorganization and formation of structures that enable cell movement. Besides having similar requirements than migration, invasion also involves proteolytic degradation of extracellular matrix (ECM). Matrix metalloproteases (MMPs) have been identified to break down components of the ECM, allowing cancer cells to spread out of the primary tumor...
July 29, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28735385/preparation-of-decellularized-liver-scaffolds-and-recellularized-liver-grafts
#18
Yibin Chen, Sharon Geerts, Maria Jaramillo, Basak E Uygun
Severe liver disease is the 12th leading cause of death in the USA, with organ transplantation often being the only viable option for treatment. However, due to the shortage of viable donor livers, it is estimated that over 1200 patients died in 2015 while waiting for liver transplantation. This highlights the need for alternative sources of viable organs. In this study, we describe a method that provides the groundwork for the development of functional liver grafts. The approach described here is for removal of cells from intact livers and subsequently repopulating them with functional liver cells...
July 23, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28730534/tracheal-cartilage-isolation-and-decellularization
#19
Zachary Galliger, Angela Panoskaltsis-Mortari
Tissue decellularization allows isolation of tissue extracellular matrix components, enabling bioengineering of native tissue microenvironments with minimal antigenic components. Here we describe a method to harvest decellularized cartilage tissue from donor trachea using a series of chemical and enzymatic washes and incorporating the extracellular matrix in gelatin methacrylate hydrogels. This decellularized cartilage tissue is easily incorporated into a variety of hydrogels to create a cartilage tissue scaffold...
July 21, 2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28710687/disaggregation-and-reaggregation-of-zebrafish-retinal-cells-for-the-analysis-of-neuronal-layering
#20
Megan K Eldred, Leila Muresan, William A Harris
The reaggregation of dissociated cells to form organotypic structures provides an in vitro system for the analysis of the cellular interactions and molecular mechanisms involved in the formation of tissue architecture. The retina, an outgrowth of the forebrain, is a precisely layered neural tissue, yet the mechanisms underlying layer formation are largely unexplored. Here we describe the protocol to dissociate, re-aggregate, and culture zebrafish retinal cells from a transgenic, Spectrum of Fates, line where all main cell types are labelled with a combination of fluorescent proteins driven by fate-specific promoters...
July 15, 2017: Methods in Molecular Biology
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