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This detailed book collects original protocols aimed at encouraging and stimulating the scientific community to design and produce models for the laboratory that mimic cell guidance conditions as they occur in vivo. The protocols collected describe powerful strategies to exploit chemical cues involved in cell differentiation processes. Special emphasis is given to the use of methods for purification and characterization of exosomes and other secreted vesicles, as well as micro and non-coding RNAs, that have been demonstrated to control the tuning of the in vivo micro and macro environment in order to ensure the optimal soluble environment in vitro. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Next Generation Culture Platforms for Reliable In Vitro Models: Methods and Protocols serves as an ideal guide for researchers working toward developing these vital testing models for preclinical studies.
List of contents
Recreating the Follicular Environment: A Customized Approach for In Vitro Culture of Bovine Oocytes Based on the Origin and Differentiation State.- Clinostat 3D Cell Culture: Protocols for the Preparation and Functional Analysis of Highly Reproducible, Large, Uniform Spheroids and Organoids.- Protocol to Study the Role of Extracellular Vesicles during Induced Stem Cell Differentiation.- A Simplified Method for Three-Dimensional (3D) Porcine Preantral Follicles Culture Utilizing Hydrophobic Microbioreactors.- Use of Transparent Liquid Marble: Microbioreactor to Culture Cardiospheres.- Isolation, Culture, and Characterization of Primary Bovine Endometrial, Epithelial, and Stromal Cells for 3D In Vitro Tissue Models.- Using Decellularization/Recellularization Processes to Prepare Liver and Cardiac Engineered Tissues.- Use of Virus-Mimicking Nanoparticles to Investigate Early Infection Events in Upper Airway 3D Models.- Creation of a Bioengineered Ovary: Isolation of Female Germline Stem Cells for the Repopulation of a Decellularized Ovarian Bio-Scaffold.- A Two-Step Protocol to Erase Human Skin Fibroblasts and Convert Them into Trophoblast-Like Cells.- 3D-ViaFlow: A Quantitative Viability Assay for Multicellular Spheroids.- Method to Disassemble Spheroids into Core and Rim for Downstream Applications Such as Flow Cytometry, Comet Assay, Transcriptomics, Proteomics, and Lipidomics.- Isolation of Extracellular Vesicles (EVs) Using Size Exclusion-High Performance Liquid Chromatography (SEC-HPLC).- Isolation of Extracellular Vesicles (EVs) Using Benchtop Size Exclusion Chromatography (SEC) Columns.- Measurement of the Size and Concentration and Zeta Potential of Extracellular Vesicles Using Nanoparticle Tracking Analyzer.- Isolation, Characterization, and MicroRNAs Analysis of Extracellular Vesicles from Bovine Oviduct and Uterine Fluids.- Biomimetic 3D-Bone Tissue Model.- Using the Air-Liquid Interface Approach to Foster Apical-Basal Polarization of Mammalian FemaleReproductive Tract Epithelia In Vitro.- Preparation of Biological Scaffolds and Primary Intestinal Epithelial Cells to Efficiently 3D Model the Fish Intestinal Mucosa.- Use of Porous Polystyrene Scaffolds to Bioengineer Human Epithelial Tissues In Vitro.
