BIOMEDICAL TISSUE CULTURE Edited by Luca Ceccherini-Nelli Barbara Matteoli BIOMEDICAL TISSUE CULTURE Edited by Luca Ceccherini-Nelli and Barbara Matteoli Biomedical Tissue Culture Edited by Luca Ceccherini-Nelli and Barbara Matteoli Contributors Zhanqiu Yang, Hai-Rong Xiong, Satoru Kaneko, Kiyoshi Takamatsu, Shlomo Rottem, Nechama S. Kosower, Jonathan D. Kornspan, Phuc Van Pham, Binh Thanh Vu, Nhan Lu Chinh Phan, Thuy Thanh Duong, Tue Gia Vuong, Giang Do Thuy Nguyen, Thiep Van Tran, Dung Xuan Pham, Minh Hoang Le, Ngoc Kim Phan, O.S. Sotnikov, Jo~ao Bosco Barreto Filho, Maira Souza Oliveira, Seiji Omata, Yoshinori Sawae, Teruo Murakami, John A. Lednicky, Diane E. Wyatt, Paulina Godzik, Aurora Longa Brice~no, Zulma Pe~na Contreras, Delsy Dávila Vera, Rosa Mendoza Brice~no, Ernesto Palacios Prü, B. Matteoli, L. Ceccherini-Nelli Copyright (c) 2016 All chapters are Open Access distributed under the Creative Commons Attribution 3.0 license, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. After this work has been published, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. First published October, 2012 second - 2016 ISBN-10: 953-51-0788-7 ISBN-13: 978-953-51-0788-0 Contents Preface IX Section 1 General Characteristics and Culture Conditions 1 Chapter 1 Culture Conditions and Types of Growth Media for Mammalian Cells 3 Zhanqiu Yang and Hai-Rong Xiong Chapter 2 Cell Handling and Culture Under Controlled Oxygen Concentration 19 Satoru Kaneko and Kiyoshi Takamatsu Chapter 3 Contamination of Tissue Cultures by Mycoplasmas 35 Shlomo Rottem, Nechama S. Kosower and Jonathan D. Kornspan Chapter 4 Isolation of Breast Cancer Stem Cells by Single-Cell Sorting 59 Phuc Van Pham, Binh Thanh Vu, Nhan Lu Chinh Phan, Thuy Thanh Duong, Tue Gia Vuong, Giang Do Thuy Nguyen, Thiep Van Tran, Dung Xuan Pham, Minh Hoang Le and Ngoc Kim Phan Chapter 5 In vitro, Tissue-Based Models as a Replacement for Animal Models in Testing of Drugs at the Preclinical Stages 73 Zhanqiu Yang and Hai-Rong Xiong Section 2 Specific Uses of Tissue Culture 81 Chapter 6 Use of Cell Culture to Prove Syncytial Connection and Fusion of Neurons 83 O.S. Sotnikov Chapter 7 Placental Structure and Biological Aspects of Fetal Membranes Cultured in vitro 115 Jo~ao Bosco Barreto Filho and Maira Souza Oliveira VI Contents Chapter 8 Tissue Development and Mechanical Property in the Regenerated-Cartilage Tissue 133 Seiji Omata, Yoshinori Sawae and Teruo Murakami Chapter 9 The Art of Animal Cell Culture for Virus Isolation 151 John A. Lednicky and Diane E. Wyatt Chapter 10 Tissue-Based Model of HCV Replication as a Replacement for Animal Models in Drug Testing 179 Paulina Godzik Chapter 11 Tissue Culture to Assess Bacterial Enteropathogenicity 203 Aurora Longa Brice~no, Zulma Pe~na Contreras, Delsy Dávila Vera, Rosa Mendoza Brice~no and Ernesto Palacios Prü Chapter 12 Viral DNA and cDNA Array in the Diagnosis of Respiratory Tract Infections 221 B. Matteoli and L. Ceccherini-Nelli Preface In this chapters many aspects of tissue culture models are extensively studied. The authors describe a coltural method to produce massive neuronal syncytial connections and induce their fusion with formation of bi- and multinucleated cells. They show the applicability in research of cell lines derived from the cultivation of placenta with the properties of progenitor/stem cells. They show the procedures that can regenerate cartilage tissue with appropriate mechanical characteristics. We report here many applications of tissue cultures in Microbiology, for virus isolation, also for those viruses once considered very difficult to study in vitro, that can be used to replace animal models for drug testing at the preclinical stages, based on tissue- models for HCV replication; in the study of pathogenicity of enteropathogenic bacteria; in the diagnosis of respiratory tract infections (RTI) with the use of viral DNA and cDNA Arrays compared with routine diagnostic methods. Prof. Luca Ceccherini-Nelli, MD Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia Università degli Studi di Pisa , Italy Dott.ssa Barbara Matteoli Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia Università degli Studi di Pisa Italy Section 1 General Characteristics and Culture Conditions Chapter 1 Culture Conditions and Types of Growth Media for Mammalian Cells Zhanqiu Yang and Hai-Rong Xiong Additional information is available at the end of the chapter dx.doi/10.5772/52301 1. Introduction 1.1. Basic requirement for culture medium 1.1.1. Nutritional components Cells need the basic nutritional conditions to grow in vitro, including: 1. Amino acid Amino acid is the raw material for the cell to synthesize protein. All the cells need twelve essential amino-acids: arginine, cystine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, histidine, tyrosine and valine, which are L-amino acids. Furthermore, glutamine is another component playing important role in the cell metabolism process. The nitrogen contained in glutamine is not only the source of purine and pyrimidine of nucleic acid, but also the essential material for the synthesis of the Tri-, bi-, mono-phosphate acid glycosides. 2. Monosaccharide Cultured cells use aerobic glycolysis and anaerobic glycolysis of hexose as main energy source. In addition, hexose is used for the synthesis of some amino acid, fat and nucleic acid. Cell absorptive capacity varies among different monosaccharides, with the highest for glucose and the lowest for galactose. 3. Vitamin Vitamins mainly act as coenzymes or prothetic groups in cell metabolism processes. Biotin, folate, nicotinamide, pantothenic acid, pyridoxine, riboflavin, thiamine and vitamin B12 are common component in culture medium. 4 Biomedical Tissue Culture 4. Inorganic ion and trace element Besides some basic elements (including sodium, potassium, calcium, magnesium, nitrogen and phosphorus), cell growth needs some trace elements, such as molybdenum, vanadium, iron, zinc and selenium, copper, manganese. 1.2. Somatomedin and hormones Cells grown in vivo are always regulated by somatomedin and hormones. Many researches demonstrate that various somatomedin and hormones are very important to maintain cell function and status (differentiated or undifferentiated). Some hormones have promoting growth effects on different cell typs. For instance, insulin can promote the use of glucose and amino acids in the cell. Some hormones are cell-type specific, as hydrocortisone that can promote the growth of epidermal cells and prolaction that induces the proliferation of mammary epithelial cell. 1.3. Osmotic pressure Cells need an isotonic environment and human plasma osmotic pressure is about 290 mOsm/kg, which is thought to be ideal osmotic pressure to culture human cells. Mouse plasma osmotic pressure is about 320 mOsm/kg. Osmotic pressure of 260-320 mOsm/kg fits for most mammalian cells. 1.4. pH The suitable pH for most cells is 7.2-7.4; otherwise it will produce harmful effects. The culture medium should have some buffer capacity. The main substance causing pH changes is CO2 produced in cell metabolism process. In an airtight environment, CO2 can combine H2O2 to produce carbonic acid and thus reduce the pH value of the medium. Synthesized medium employs NaHCO3-CO2 buffer system to solve this problem. In the buffer system, the boost in [H+] increases the reaction rate H+ + salt => weak acid and takes some H+ out of circulation. It is based on the constant equilibrium. +←→+++↑+- NaHCO3 H222 O Na HO H O CO 2. Natural medium Natural medium is described as animal body fluids or medium of tissue extraction, including plasma, serum, lymph, chicken embryos leaching solution. Natural medium contains rich nutrients, various somatomedin and hormones, similar osmotic pressure and pH to body environment. As this medium has a very complicated production process and big batch-to-batch variation, the medium is gradually replaced by the synthetic med