Next Generation Culture Platforms for Reliable In Vitro Models Methods and Protocols

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. 

1. Recreating the Follicular Environment: A Customized Approach for In Vitro Culture of Bovine Oocytes Based on the Origin and Differentiation State

            Alberto Maria Luciano, Rodrigo Garcia Barros, Ana Caroline Silva Soares, Jose Buratini, Valentina Lodde, and Federica Franciosi

 

2. Clinostat 3D Cell Culture: Protocols for the Preparation and Functional Analysis of Highly Reproducible, Large, Uniform Spheroids and Organoids

            Krzysztof Wrzesinski, Helle Sedighi Frandsen, Carlemi Calitz, Chrisna Gouws, Barbara Korzeniowska, and Stephen J. Fey

 

3. Protocol to Study the Role of Extracellular Vesicles during Induced Stem Cell Differentiation

            Kelly C.S. Roballo, Carlos E. Ambrosio, and Juliano C. da Silveira

 

4. A Simplified Method for Three-Dimensional (3D) Porcine Preantral Follicles Culture Utilizing Hydrophobic Microbioreactors

            Malgorzata Duda , Lucia Gizler, and Gabriela Gorczyca

 

5. Use of Transparent Liquid Marble: Microbioreactor to Culture Cardiospheres     

            Jeffrey Aalders, Laurens Léger, Davide Piras, Jolanda van Hengel, and Sergio Ledda

 

6. Isolation, Culture, and Characterization of Primary Bovine Endometrial, Epithelial, and Stromal Cells for 3D In Vitro Tissue Models

            Antonio Murillo and Marta Muñoz

 

7. Using Decellularization/Recellularization Processes to Prepare Liver and Cardiac Engineered Tissues

            Matteo Ghiringhelli, Yousef Abboud, Snizhanna V. Chorna, Irit Huber, Gil Arbel, Amira Gepstein, Georgia Pennarossa, Tiziana A.L. Brevini, and Lior Gepstein

 

8. Use of Virus-Mimicking Nanoparticles to Investigate Early Infection Events in Upper Airway 3D Models

            Georgia Pennarossa, Alireza Fazeli, Sergio Ledda, Fulvio Gandolfi, and Tiziana A.L. Brevini

 

9. Creation of a Bioengineered Ovary: Isolation of Female Germline Stem Cells for the Repopulation of a Decellularized Ovarian Bio-Scaffold

            Georgia Pennarossa, Matteo Ghiringhelli, Fulvio Gandolfi, and Tiziana A.L. Brevini

 

10. A Two-Step Protocol to Erase Human Skin Fibroblasts and Convert Them into Trophoblast-Like Cells

            Sharon Arcuri, Fulvio Gandolfi, Edgardo Somigliana, and Tiziana A.L. Brevini

 

11. 3D-ViaFlow: A Quantitative Viability Assay for Multicellular Spheroids

            Joel Mario Vej-Nielsen and Adelina Rogowska-Wrzesinska

 

12. Method to Disassemble Spheroids into Core and Rim for Downstream Applications Such as Flow Cytometry, Comet Assay, Transcriptomics, Proteomics, and Lipidomics

            Helle Sedighi Frandsen, Martina Štampar, Joel Mario Vej-Nielsen, Bojana Žegura, and Adelina Rogowska-Wrzesinska

 

13. Isolation of Extracellular Vesicles (EVs) Using Size Exclusion-High Performance Liquid Chromatography (SEC-HPLC)

            Keerthie Dissanayake, Kasun Godakumara, and Alireza Fazeli

 

14. Isolation of Extracellular Vesicles (EVs) Using Benchtop Size Exclusion Chromatography (SEC) Columns

            Qurat Ul Ain Reshi, Mohammad Mehedi Hasan, Keerthie Dissanayake, and Alireza Fazeli

 

15. Measurement of the Size and Concentration and Zeta Potential of Extracellular Vesicles Using Nanoparticle Tracking Analyzer

            Keerthie Dissanayake, Getnet Midekessa, Freddy Lättekivi, and Alireza Fazeli

 

16. Isolation, Characterization, and MicroRNAs Analysis of Extracellular Vesicles from Bovine Oviduct and Uterine Fluids

            Karina Cañón-Beltrán, Meriem Hamdi, Rosane Mazzarella, Yulia N. Cajas, Claudia L.V. Leal, Alfonso Gutiérrez-Adán, Encina M. González, Juliano C. da Silveira, and Dimitrios Rizos

 

17. Biomimetic 3D-Bone Tissue Model

            Mahmut Parmaksiz, Ayse Eser Elçin, and Yasar Murat Elçin

 

18. Using the Air-Liquid Interface Approach to Foster Apical-Basal Polarization of Mammalian Female Reproductive Tract Epithelia In Vitro

            Shuai Chen and Jennifer Schoen

 

19. Preparation of Biological Scaffolds and Primary Intestinal Epithelial Cells to Efficiently 3D Model the Fish Intestinal Mucosa

            Nicole Verdile, Anna Szabo, Rolando Pasquariello, Tiziana A.L. Brevini, Sandra Van Vlierberghe, and Fulvio Gandolfi

 

20. Use of Porous Polystyrene Scaffolds to Bioengineer Human Epithelial Tissues In Vitro

            Lydia Costello, Nicole Darling, Matthew Freer, Steven Bradbury, Claire Mobbs, and Stefan Przyborski