Introduction to Cell and Tissue Culture Theory and Technique huangzhiman 2002.12.18 Foreword Chapter 1: Introduction 1 Chapter 2: Setting Up a Cell Culture Laboratory 9 Chapter 3: The Physical Environment 25 Chapter 4: Media 41 Chapter 5: Standard Cell Culture Techniques 63 Chapter 6: Looking at Cells 89 Chapter 7: Contamination: How to Avoid It, Recognize It, and Get Rid of It 117 Chapter 8: Serum-Free Culture 129 Chapter 9: Primary Cultures 151 Chapter 10: Establishing a Cell Line 165 Chapter 11: Special Growth Conditions 175 Chapter 12: Cell Culture for Commercial Settings 195 Glossary Appendix Index 205 Page i Introduction to Cell and Tissue Culture Theory and Technique Page ii INTRODUCTORY CELL AND MOLECULAR BIOLOGY TECHNIQUES SERIES EDITOR: Bonnie S. Dunbar, Baylor College of Medicine, Houston, Texas INTRODUCTION TO CELL AND TISSUE CULTURE: Theory and Technique Jennie P. Mather and Penelope E. Roberts Page iii Introduction to Cell and Tissue Culture Theory and Technique Jennie P. Mather and Penelope E. Roberts Genentech Inc. South San Francisco, California Page iv Library of Congress Cataloging-in-Publication Data Mather, Jennie P., 1948¨C Introduction to cell and tissue culture : theory and technique / Jennie P. Mather and Penelope E. Roberts. p. cm.¡ª (Introductory cell and molecular biology techniques) Includes bibliographical references and index. ISBN 0-306-45859-4 1. Cell culture. 2. Tissue culture. I. Roberts, Penelope E. II. Title. III. Series. [DNLM: 1. Tissue Culture¡ªmethods. 2. Microbiological Techniques. QS 525M427i 1998] QH585.2.M38 1998 571.5'38¡ªdc21 DNLM/DLC for Library of Congress 98-27597 CIP ISBN 0-306-45859-4 ©1998 Plenum Press, New York A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013 http://www.plenum.com 10 9 8 7 6 5 4 3 2 1 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher Printed in the United States of America Page v To the memory of Dr. Izumi Hayashi, whose life was as elegant as her experiments Page vii Foreword It is a pleasure to contribute the foreword to Introduction to Cell and Tissue Culture: Theory and Techniques by Mather and Roberts. Despite the occasional appearance of thoughtful works devoted to elementary or advanced cell culture methodology, a place remains for a comprehensive and definitive volume that can be used to advantage by both the novice and the expert in the field. In this book, Mather and Roberts present the relevant methodology within a conceptual framework of cell biology, genetics, nutrition, endocrinology, and physiology that renders technical cell culture information in a comprehensive, logical format. This allows topics to be presented with an emphasis on troubleshooting problems from a basis of understanding the underlying theory. The material is presented in a way that is adaptable to student use in formal courses; it also should be functional when used on a daily basis by professional cell culturists in academia and industry. The volume includes references to relevant Internet sites and other useful sources of information. In addition to the fundamentals, attention is also given to modem Introduction to Culture Introduction to Culture Bởi: OpenStaxCollege Graffiti’s mix of colorful drawings, words, and symbols is a vibrant expression of culture—or, depending on one’s viewpoint, a disturbing expression of the creator’s lack of respect for a community’s shared space (Photo courtesy of aikijuanma/flickr) Are there rules for eating at McDonald’s? Generally, we not think about rules in a fast food restaurant, but if you look around one on a typical weekday, you will see people acting as if they were trained for the role of fast food customer They stand in line, pick items from the colorful menus, swipe debit cards to pay, and wait to collect trays of food After a quick meal, customers wad up their paper wrappers and toss them into garbage cans Customers’ movement through this fast food routine is orderly and predictable, even if no rules are posted and no officials direct the process If you want more insight into these unwritten rules, think about what would happen if you behaved according to some other standards (You would be doing what sociologists call ethnomethodology: deliberately disrupting social norms in order to learn about them.) For example, call ahead for reservations, ask the cashier detailed questions about the food’s ingredients or how it is prepared Ask to have your meal served to you at your table Or throw your trash on the ground as you leave Chances are, you will elicit hostile responses from the restaurant employees and your fellow customers 1/2 Introduction to Culture People have written entire books analyzing the significance of fast food customs They examine the extensive, detailed physicality of fast food: the food itself, wrappers, bags, trays, those tiny ketchup packets, the tables and chairs, and even the restaurant building Everything about a chain restaurant reflects culture, the beliefs and behaviors that a social group shares Sociological analysis can be applied to every expression of culture, from sporting events to holidays, from education to transportation, from fashion to etiquette In everyday conversation, people rarely distinguish between the terms culture and society, but the terms have slightly different meanings, and the distinction is important to a sociologist A society describes a group of people who share a community and a culture By “community,” sociologists refer to a definable region—as small as a neighborhood (Brooklyn, or “the east side of town”), as large as a country (Ethiopia, the United States, or Nepal), or somewhere in between (in America, this might include someone who identifies with Southern or Midwestern society) To clarify, a culture represents the beliefs and practices of a group, while society represents the people who share those beliefs and practices Neither society nor culture could exist without the other In this chapter, we examine the relationship between culture and society in greater detail, paying special attention to the elements and forces that shape culture, including diversity and cultural changes A final discussion touches on the different theoretical perspectives from which sociologists research culture 2/2 American Ways An Introduction to American Culture Fourth Edition Teacher’s Manual with Answer Key Online Maryanne Kearny Datesman JoAnn Crandall Edward N Kearny American Ways: An Introduction to American Culture, Fourth Edition Teacher’s Manual with Answer Key Online Copyright © 2014 by Pearson Education, Inc All rights reserved No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior permission of the publisher Pearson Education, 10 Bank Street, White Plains, NY 10606 Staff Credits: The people who made up the American Ways, Fourth Edition, Teacher’s Manual team are Lisa Bayrasli, Barbara Del Rowe, Warren Fischbach, Amy McCormick, Joan Poole, and Debbie Sistino ISBN: 978-0-13-304703-5; 0-13-304703-2 ii Contents PART 1: HOW TO USE THIS MANUAL Teaching American Culture in the Language Classroom How to Use the Activities in the Text Before You Read After You Read Skill Building Expand Your Knowledge Write About It Explore on Your Own PART 2: ANSWER KEY FOR STUDENT TEXT Chapters 1–12 PART 3: REPRODUCIBLE ACTIVITY MASTERS 1 14 15 17 17 39 iii PART HOW TO USE THIS MANUAL Teaching American Culture in the Language Classroom Why Include Culture in Language Classes? There are a number of reasons for including culture in your language classes: • Culture provides interesting content for language learning, leading to engaging discussions, writing assignments, or group projects, and also can be used to develop both informal social language and more formal academic language • Culture can also be used to increase the cognitive component of the language class, helping students to develop higher order thinking skills as they analyze, compare, and discuss the cultural content • Reading about and discussing other cultures can serve as a valuable backdrop for analyzing students’ own cultures It is often said that we not really understand our own culture until we have lived outside it, or seen it through another person’s eyes In the words of a famous proverb, “A fish that never leaves the water does not discover water.” • Studying culture can lead to a better understanding of people’s behavior and help students move from ethnocentric pronouncements of what is “right” or “wrong” to more thoughtful tolerance of cultural diversity Culture, then, can be interesting content, even for those students whose primary motivation for learning English is academic, for it promotes complex linguistic and cognitive interaction and encourages students to use the kinds of skills and language that are required for both academic and professional contexts What Culture Should Be Taught? Traditional definitions of culture—or what has been called “culture with a capital C”—focus on the literature, music, dance, drama, and other arts of a group or a country That is often the focus of cultural studies in traditional language classrooms, especially the study of literature as a window to that culture Although this “Culture” is important, what may be of more interest to students and potentially more useful to them, if they are going to interact with people from a new culture, is to understand culture as it is more commonly understood by anthropologists: that is, the set of beliefs, attitudes, and behaviors or customs that define or distinguish a group of people Or as Edward T Hall defines it, “a set of ideals, values, and standards of behavior that make the actions of individuals intelligible to the group.” In this book, culture represents the ways of perceiving, thinking, communicating, behaving, and evaluating that characterize Americans Culture is shared, but in a country as large and diverse as the United States, there is also a great deal of cultural diversity based on ethnicity, race, gender, and/or social class, all of which create a number of different societies in the country But even with these differences, there is still Introduction to Animal Tissue culture What is tissue culture?  In vitro culture (maintain and/or proliferate) of cells, tissues or organs  Types of tissue culture • Cell culture • Primary explant culture • Organ culture Three major categories of tissue culture  Cell culture: Adherent monolayer on a solid substrate (various cell types) suspension in the culture medium (few cell types)  Primary explant culture: A fragment of tissue attachment and migration occurs in the plane of the solid substrate Expla Expla  nt nt Organ culture: cultur cultur ee A spherical or three-dimensional shape specific histological interaction Cell Cell cult cult ure ure Explant: living cells, tissues, or organs from animals or plants that transfer to a nutrient medium Cell culture & Enzymatic Dissociation Tissue from an explant is dispersed, mostly enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture Advantages & Disadvantages  Advantages  Development of a cell line over several generations  Scale-up is possible  Absolute control of physical environment  Homogeneity of sample  Less compound needed than in animal models  Disadvantages  Cells may lose some differentiated characteristics  Hard to maintain  Only grow small amount of tissue at high cost  Dedifferentiation  Instability, aneuploidy Tissue Culture  Is the growth of tissues or cells separate from the organism  This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar Advantages & Disadvantages  Advantages   Some normal functions may be maintained Better than organ culture for scale-up but not ideal  Disadvantages  Original organization of tissue is lost Organ culture  The entire embryos or organs are excised from the body and culture  Advantages Normal physiological functions are maintained Cells remain fully differentiated  Disadvantages Scale-up is not recommended Growth is slow Fresh explantation is required for every experiment      EMP04 Why we need Cell culture?  Research ◦ To overcome problems in studying cellular behavior such as:   ◦  confounding effects of the surrounding tissues variations that might arise in animals under experimental stress Reduce animal use Commercial or large-scale production ◦ Production of cell material: vaccine, antibody, hormone 10 Initiation of culture Animal Plant Tissue Primary Primary culture culture Subculture Stored Stored Cell line Finite numbers Continuous cell line Indefinite numbers 11 Types of Cell culture Primar y Culture Preparation Primary Cultures .Derived directly from excised tissue and cultured either as: .Outgrowth of excised tissue in culture .Dissociation into single cells (by enzymatic digestion or mechanical dispersion) 12 Characteristics of Primary Cultures Primar y Culture  Characteristics:  Morphologically similar to the parent tissue  Limited number of cell divisions  Best experimental models for in vivo situations Preparation 13 Advantages & Disadvantages ◦ Advantages:  ◦ usually retain many of the differentiated characteristics of the cell in vivo Disadvantages:        initially heterogeneous but later become dominated by fibroblasts the preparation of primary cultures is labor intensive can be maintained in vitro only for a limited period of time Difficult to obtain Relatively short life span in culture Very susceptible to contamination May not fully act like tissue due to complexity of media 14 Types of Cell culture Continuous Cultures    derived from subculture (or passage, or transfer) of primary culture  Subculture = the process of dispersion and re-culture the cells after they have increased to occupy all of the available substrate in the culture usually comprised of a single cell type can be serially propagated in culture for several passages  There are two types of continuous cultures  Cell lines  Continuous cell lines 15 Types of continuous culture 1) Cell lines  ... reflects culture, the beliefs and behaviors that a social group shares Sociological analysis can be applied to every expression of culture, from sporting events to holidays, from education to transportation,.. .Introduction to Culture People have written entire books analyzing the significance of fast food customs They examine the extensive, detailed physicality... relationship between culture and society in greater detail, paying special attention to the elements and forces that shape culture, including diversity and cultural changes A final discussion touches on