Culture of animal cells : a manual of basic technique

©WILE Y

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A

ultur e o

nim a i l G e

MANUA L O F BASI C TECHNIQ U

A MANUA L

OF BASIC TECHNIQUE

Fifth Edition

R . Ia n Freshne y

Cancer Research U K Centre for Oncology and Applied Pharmacology

Cancer Research U K Beatson Laboratories

University o f Glasgow

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©WILEY-LIS S

A JOHN WILEY & SONS, INC., PUBLICATION

Cover photographs courtesy of Hans-Jiirgen Stark and Hee-Young Park (see Plates 8d and 11a for details)

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Library of Congress Cataloging-in-Publication Data:

Freshney, R. Ian.

Culture of animal cells : a manual of basic techmques/R. Ian Freshney.—5th ed. p. cm.

Includes bibliographical references and index.

ISBN-13 978-0-471-45329-1 (alk. paper)

ISBN-10 0-471-45329-3 (alk. paper)

1. Tissue culture Laboratory manuals. 2. Cell culture Laboratory manuals. I. Title.

QH585.2.F74 1994

571.6'38—dc21 99-23536

Pnnted in the United States of Amenca.

10 9876543

This book is dedicated to all o f the many friends and colleagues whose help and advice

over the years has enabled me to extend the scope o f this book beyond

my own limited experience.

••••••••••••••••••••••••••••••••••• •

n

List of Figures, xvii

List of Color Plates, xxi

Preface, xxiii

Abbreviations, xxv

1. Introduction, 1

1.1. Historical Background, 1

1.2. Advantages of Tissue Culture, 6

1.2.1. Control of the Environment, 6

1.2.2. Characterization and Homogeneity of

Sample, 6

1.2.3. Economy, Scale, and Mechanization, 6

1.2.4. In Vitro Modeling of In Vivo Conditions, 7

1.3. Limitations, 7

1.3.1. Expertise, 7

1.3.2. Quantity, 7

1.3.3. Dedifferentiation and Selection, 7

1.3.4. Origin of Cells, 7

1.3.5. Instability, 7

1.4. Major Differences In Vitro, 7

1.5. Types of Tissue Culture, 8

I Training Programs, 11

2.1. Objectives, 11

2.2. Basic Exercises, 11

Exercise 1 Aseptic Technique I: Pipetting and Transfer

of Fluids, 13

Exercise 2 Introduction to Cell Cultures, 14

S

Exercise 3 Aseptic Technique IT. Preparing Medium for

Use, 15

Exercise 4 Feeding a Monolayer Culture, 15

Exercise 5 Washing and Sterilizing Glassware, 16

Exercise 6 Preparation and Sterilization of Water, 16

Exercise 7 Preparation and Sterilization of Dulbecco's

Phosphate-Buffered Saline (D-PBS) without Ca2+ and

Mg2

* (D-PBSA), 17

Exercise 8 Preparation of pH Standards, 17

Exercise 9 Preparation of Stock Medium from Powder

and Sterilization by Filtration, 18

Exercise 10 Preparation of Complete Medium from

Wx Stock, 18

Exercise 11 Preparation of Complete Medium from

Powder, 19

Exercise 12 Counting Cells by Hemacytometer and

Electronic Counter, 19

Exercise 13 Subculture of Cells Growing in

Suspension, 20

Exercise 14 Subculture of Continuous Cell Line

Growing in Monolayer, 22

Exercise 15 Staining a Monolayer Cell Culture with

Giemsa, 23

Exercise 16 Construction and Analysis of Growth

Curve, 23

2.3. Advanced Exercises, 24

Exercise 17 Cryopreservation of Cultured Cells, 25

Exercise 18 Detection of Mycoplasma, 25

Exercise 19 Cell Line Characterization, 27

Exercise 20 Primary Culture, 28

Exercise 21 Cloning of Monolayer Cells, 29

2.4. Specialized Exercises, 30

vii

viii *# CONTENTS

3. Biology of Cultured Cells, 31

3.1. The Culture Environment, 31

3.2. Cell Adhesion, 31

3.2.1. Cell Adhesion Molecules, 31

3.2.2. Intercellular Junctions, 32

3.2.3. Extracellular Matrix, 33

3.2.4. Cytoskeleton, 33

3.2.5. Cell Motility, 34

3.3. Cell Proliferation, 34

3.3.1. Cell Cycle, 34

3.3.2. Control of Cell Proliferation, 35

3.4. Differentiation, 35

3.4.1. Maintenance of Differentiation, 35

3.4.2. Dedifferentiation, 37

3.5. Cell Signaling, 38

3.6. Energy Metabolism, 39

3.7. Initiation of the Culture, 39

3.8. Evolution of Cell Lines, 40

3.8.1. Senescence, 41

3.9. The Development of Continuous Cell Lines, 41

3.10. Origin of Cultured Cells, 42

4. Laboratory Design and Layout, 43

4.1. Planning, 43

4.2. Construction and Services, 44

4.3. Layout of Aseptic Room or Suite, 47

4.3.1. Sterile Handling Area, 48

4.3.2. Laminar Flow, 48

4.3.3. Quarantine and Containment, 48

4.3.4. Service Bench, 48

4.4. Incubation, 48

4.4.1. Incubators, 48

4.4.2. Hot Room, 48

4.5. Preparation Area, 51

4.5.1. Media Preparation, 51

4.5.2. Washup, 51

4.5.3. Storage, 51

5. Equipment, 55

5.1. Requirements of A Tissue Culture

Laboratory, 55

5.2. Aseptic Area, 55

5.2.1. Laminar-Flow Hood, 55

5.2.2. Pipette Cylinders, 56

5.2.3. Aspiration Pump, 57

5.2.4. Service Carts, 57

5.2.5. Inverted Microscope, 58

5.2.6. Centrifuge, 58

5.2.7. Sterile Liquid Handling—Pipetting and

Dispensing, 58

5.2.8. Cell Counter, 61

5.2.9. CCD Camera and Monitor, 62

5.2.10. Dissecting Microscope, 63

5.3. Incubation, 63

5.3.1. Incubator, 63

5.3.2. Humid C0 2

Incubator, 63

5.3.3. Temperature Recorder, 64

5.3.4. Roller Racks, 64

5.3.5. Magnetic Stirrer, 65

5.4. Preparation and Sterilization, 65

5.4.1. Washup, 65

5.4.2. Water Punfier, 65

5.4.3. Sterilizing and Drying Ovens, 66

5.4.4. Steam Sterilizer (Autoclave), 66

5.4.5. Balances, 68

5.4.6. pH Meter, 68

5.4.7. Hot Plate Magnetic Stirrer, 68

5.4.8. Automatic Dispensers, 68

5.4.9. Conductivity Meter, 68

5.4.10. Osmometer, 68

5.4.11. Glassware Washing Machine, 68

5.5. Storage, 69

5.5.1. Refrigerators and Freezers, 69

5.5.2. Cryostorage Containers, 69

5.5.3. Controlled-Rate Freezer, 70

5.6. Laboratory Backup, 70

5.6.1. Computers and Networks, 70

5.6.2. Upright Microscope, 70

5.6.3. Low-Temperature Freezer, 70

5.6.4. Confocal Microscope, 70

5.6.5. PCR Cycler, 71

5.7. Specialized Equipment, 71

5.7.1. Microinjection Facilities, 71

5.7.2. Colony Counter, 71

5.7.3. Centrifugal Elutriator, 71

5.7.4. Flow Cytometer, 71

5.8. Consumable Items, 71

5.8.1. Pipettes, 71

5.8.2. Hemocytometer, 72

5.8.3. Culture Vessels, 72

5.8.4. Sterile Containers, 72

5.8.5. Syringes and Needles, 72

5.8.6. Sterilization Filters, 72

5.8.7. Paper Towels and Swabs, 72

5.8.8. Disinfectants, 72

G. Aseptic Technique, 73

6.1. Objectives of Aseptic Technique, 73

6.1.1. Maintaining Sterility, 73

6.2. Elements of Aseptic Environment, 74

6.2.1. Quiet Area, 74

6.2.2. Work Surface, 74

6.2.3. Personal Hygiene, 76

6.2.4. Reagents and Media, 76

6.2.5. Cultures, 76