Animal Cell Culture E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 652

Veröffentlichungsjahr: 2011

Beschreibung

This is a comprehensive research guide that describes both the key new techniques and more established methods. Every chapter discusses the merits and limitations of the various approaches and then provides selected tried-and-tested protocols, as well as a plethora of good practical advice, for immediate use at the bench. It presents the most accessible and comprehensive introduction available to the culture and experimental manipulation of animal cells. Detailed protocols for a wide variety of methods provide the core of each chapter, making new methodology easily accessible.
This book is an essential laboratory manual for all undergraduates and graduates about to embark on a cell culture project. It is a book which both experienced researchers and those new to the field will find invaluable.

Leseprobe

Contents

Cover

Title Page

Copyright

Dedication

Contributors

preface

Abbreviations

Protocols

1 The Cell Culture Laboratory

1.1 Introduction

1.2 Methods and approaches

1.3 Troubleshooting

Acknowledgements

2: Sterilization

2.1 Introduction

2.2 Methods and approaches

2.3 Troubleshooting

3: Microscopy of Living Cells

3.1 Introduction

3.2 Methods and approaches

3.3 Troubleshooting

Acknowledgements

4: Basic Techniques and Media, the Maintenance of Cell Lines, and Safety

4.1 Introduction

4.2 Methods and approaches

4.3 Troubleshooting

Acknowledgements

5: Development and Optimization of Serum- and Protein-free Culture Media

5.1 Introduction

5.2 Methods and approaches

5.3 Troubleshooting

5.4 Conclusion

Acknowledgments

6: Cryopreservation and Banking of Cell Lines

6.1 Introduction

6.2 Methods and approaches

6.3 Troubleshooting

Acknowledgement

7: Primary Culture of Specific Cell Types and the Establishment of Cell Lines

7.1 Introduction

7.2 Methods and approaches

7.3 Troubleshooting

8: Cloning

8.1 Introduction

8.2 Methods and approaches

8.3 Troubleshooting

9: The Quality Control of Animal Cell Lines and the Prevention, Detection and Cure of Contamination

9.1 Introduction

9.2 Methods and approaches

9.3 Troubleshooting

10: Systems for Cell Culture Scale-up

10.1 Introduction

10.2 Methods and approaches

10.3 Troubleshooting

11: Good Laboratory Practice in the Cell Culture Laboratory

11.1 Introduction

11.2 Background to GLP

11.3 General GLP principles

11.4 Study performance

11.5 Good Manufacturing Practice

11.6 Summary

Color Plate

Index

This edition first published 2011, © 2011 by John Wiley & Sons Ltd

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Library of Congress Cataloging-in-Publication Data

Animal cell culture : essential methods / editor John M. Davis. p. cm. Includes index. ISBN 978-0-470-66658-6 (pbk.) 1. Cell culture-Technique. 2. Tissue culture-Technique. I. Davis, John, 1952- QH585.2.A565 2011 571.6′381-dc22 2010037008

A catalogue record for this book is available from the British Library. This book is published in the following formats: ePDF: 978-0-470-66982-2; Wiley Online Library: 978-0-470-66981-5; ePub: 978-0-470-97563-3

For Carole, Tim and Helen, with love, as always and In memory of Ian Pearce, who often enquired about the progress of this book, but never saw it completed – thank you for the music

Contributors

Mohan Chothirakottu Abraham

John Stroger Hospital of Cook County, Chicago, IL 60612, USA

John Clarke

Haemophilia Centre, St Thomas’ Hospital, Westminster Bridge Road, London SE1 7EH, UK.

Sue Clarke

ImmunoBiology Limited, Babraham Research Campus, Babraham, Cambridge, CB22 3AT, UK

John M. Davis

School of Life Sciences, University of Hertfordshire, College Lane, Hatfield, Hertfordshire, AL10 9AB, UK

Janette Dillon

MedImmune, Granta Park, Cambridge, CB21 6GH, UK

Roland A. Fleck

National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Hertfordshire, EN6 3QG, UK

Stephen F. Gorfien

Cell Systems Division, Life Technologies Corporation, 3175 Staley Road, Grand Island, NY 14072, USA

Colin Gray

School of Medicine and Biomedical Sciences, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK

Jennifer Halsall

Eden Biodesign, Speke Road, Liverpool, L24 8RB, UK

Ross Hawkins

National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Hertfordshire, EN6 3QG, UK

David W. Jayme

Department of Biochemistry and Physical Sciences, Brigham Young University – Hawaii, 55-220 Kulanui Street #1967, Laie, HI 96762–1294, USA

David Tai Wei Leong

Cancer Science Institute, National University of Singapore, CeLS, 28 Medical Drive, Singapore 117456

Chris Morris

Health Protection Agency Culture Collections, Centre for Emergency Preparedness and Response, Porton Down, Wiltshire, SP4 0JG, UK

Kee Woei Ng

Division of Materials Technology, School of Materials Science & Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798

arbara Orton

Quality Assurance, Bio-Products Laboratory, Dagger Lane, Elstree, Hertfordshire, WD6 3BX, UK

Alison Porter

Lonza Biologics plc, Bath Road, Slough, Berkshire, SL1 4DX, UK

Peter L. Roberts

Research & Development Department, Bio-Products Laboratory, Dagger Lane, Elstree, Hertfordshire, WD6 3BX, UK

Cathy Rowe

European Collection of Cell Cultures, Porton Down, Wiltshire, SP4 0JG, UK

Jan-Thorsten Schantz

Department of Plastic and Hand Surgery, Klinikum rechts der Isar der Technischen Universita¨t Mu¨nchen, Ismaningerstrasse 22, 81675 Munich, Germany

Glyn N. Stacey

National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Hertfordshire, EN6 3QG, UK

Peter Thraves

European Collection of Cell Cultures, Porton Down, Wiltshire, SP4 0JG, UK

Daniel Zicha

Cancer Research UK, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK

Preface

This book brings together what I have found – from 37 years using mammalian cells in vitro in both academia and industry – to be the core concepts and techniques for success in the performance of cell culture, whether it be used to perform research, to generate and use test or model systems, or to produce biopharmaceuticals or other products.

Animal Cell Culture: Essential Methods is a successor to the second edition of the highly successful Basic Cell Culture: A Practical Approach [1]. However, the change of both title and publisher has provided the scope to do far more than simply revise and update an existing text. While all the basics are still here, coverage has been widened by the addition of dedicated chapters on cryopreservation, on the design and use of serum-and protein-free media, and on systems for culture scale-up.

Animal (including human) cell culture is becoming ever more important, not only in academic research (e.g. stem cells, regenerative medicine) but also in industry (e.g. for the production of monoclonal antibodies, vaccines and other products, and for toxicological testing). Thus an increasing number of scientists, technicians and students will be requiring the grounding of knowledge and skills encapsulated in this book. Similarly, the growing use of cell culture within a regulatory environment, such as in the toxicity or safety testing of pharmaceuticals, cosmetics and general chemicals, means that more and more individuals will need to work within the framework of Good Laboratory Practice, which is addressed in the final chapter of this book.

Thus it is my hope that not only will all those new to cell culture find this book a useful and concise introduction to the field, but that experienced workers will also find it of continuing use as a handy source of reference to all those tips, techniques and approaches that are at the heart of cell culture and keep the laboratory running smoothly.

Finally, my unreserved thanks go to all the authors who contributed chapters to this book. Without their unstinting efforts in sharing their considerable expertise, this book would never have moved from concept to reality.

John M. DavisHatfield, October 2010

References

Davis, J. (ed.) (2002) Basic Cell Culture: A Practical Approach, 2nd edn, Oxford University Press, Oxford, UK.

Abbreviations

2D

two-dimensional

3D

three-dimensional

ACDP

Advisory Committee on Dangerous Pathogens (UK HSE)

ATCC

American Type Culture Collection

AMV

avian myeloblastosis virus

B19

parvovirus B19 (also known as erythrovirus B19)

BSA

bovine serum albumin

BSE

bovine spongiform encephalopathy

BSI

British Standards Institution

BSS

balanced salt solution

BUN

blood urea nitrogen

BVDV

bovine viral diarrhoea virus

CCD

charge-coupled device (camera) or central composite design (statistical technique)

CDM

chemically defined media

CFE

colony-forming efficiency

cfu

colony-forming units

CFR

Code of Federal Regulations (United States)

CHO

Chinese hamster ovary (cell line)

CJD

Creutzfeld–Jakob disease

CMF-PBS

Ca2+- and Mg2+-free phosphate-buffered saline

CMF-DPBS

Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline

CM+PS

complete medium plus penicillin and streptomycin

C of A

certificate of analysis

CPA

cryoprotective agent

cpm

counts per minute (radioactivity)

DAPI

4′,6-diamidino-2-phenylindole

DIC

differential interference contrast

DMEM

Dulbecco’s modified Eagle’s medium

DMF

dimethyl formamide

DMSO

dimethylsulphoxide

DNA

deoxyribonucleic acid

DNAse

deoxyribonuclease

DO

dissolved oxygen

DOE

design of experiments (statistical analysis)

DPBS

Dulbecco’s PBS

DPBSA

Dulbecco’s PBS A

DSMZ

Deutsche Sammlung von Mikroorganismen und Zellkulturen (German culture collection)

EBSS

Earle’s balanced salt solution

ECACC

European Collection of Cell Cultures (Porton Down, UK)

ECM

extracellular matrix

EDTA

ethylenediaminetetraacetic acid

EGF

epidermal growth factor

EHS

Englebreth–Holm–Swarm (tumour)

ELISA

enzyme-linked immunosorbent assay

EMA

European Medicines Agency

EMC

encephalomyocarditis virus

EMEA

European Medicines Evaluation Agency, now renamed as the European Medicines Agency

EMEM

Eagle’s minimal essential medium

ESSS

Earle’s spinner salt solution

FACS

fluorescence-activated cell sorting

FBS

fetal bovine serum (= fetal calf serum)

FDA

United States Food and Drug Administration

FEP

fluorinated ethylene propylene (for most purposes, synonymous with PTFE)

FGF

fibroblast growth factor

FLAP

fluorescence localization after photobleaching

FRAP

fluorescence recovery after photobleaching

FRET

fluorescence resonance energy transfer

GCCP

good cell culture practice

GGT

gamma-glutamyl transferase

GLP

Good Laboratory Practice

GMEM

Glasgow minimal essential medium

GMO

genetically modified organism

GMP

Good Manufacturing Practice

HAV

hepatitis A virus

HBSS

Hanks’ balanced salt solution

HCV

hepatitis C virus

HEPA

high efficiency particulate air (filters)

HEPES

Af-(2-hydroxyethyl) piperazine-iV-(2-ethanesulphonic acid)

hES

human embryonic stem (cells)

HIV

human immunodeficiency virus

HMC

Hoffman modulation contrast

HPA

UK Health Protection Agency

HPLC

high-performance (or high-pressure) liquid chromatography

HSE

UK Health and Safety Executive

HTLV

human T-lymphotropic virus(es)

HUVECs

human umbilical vein endothelial cells

HVAC

heating, ventilation and air conditioning

IATA

International Air Transport Association

IBR

see IBRV

IBRV

infectious bovine rhinotracheitis virus

ICH

International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use

IGF

insulin-like growth factor

IMDM

Iscove’s modified Dulbecco’s medium

IPA

isopropyl alcohol (= propan-2-ol)

iPS

induced pluripotent stem cells

IRM

interference reflection microscopy

IT

information technology

IVCD

integral viable cell density

JMEM

Joklik’s minimal essential medium

JPEG

Joint Photographic Experts Group (format)

LAL

Limulus amoebocyte lysate

LDH

lactate dehydrogenase

LFC

laminar flow cabinet

LN

liquid nitrogen

LZW

Lempel-Ziv-Welch (compression)

MAD

Mutual Acceptance of Data (agreement)

MBA

microbioreactor array

MCB

master cell bank

MEM

minimal essential medium

MHRA

Medicines and Healthcare products Regulatory Agency (UK)

MLV

murine leukaemia virus

MoMLV

Moloney murine leukaemia virus

mRNA

messenger RNA

MSC

microbiological safety cabinet (= biological safety cabinet in the USA)

MTS

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt

MTT

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; this chemical is also known as thiazolyl blue tetrazolium bromide

MVM

minute virus of mice

NA

numerical aperture

NCTC

National Collection of Type Cultures (Porton Down, UK)

NIBSC

National Institute for Biological Standards and Control (South Mimms, UK)

OECD

Organization for Economic Cooperation and Development

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

PDL

population doubling level

PDT

population doubling time

PEG

polyethylene glycol

PES

polyethersulphone

PET

poly(ethylene) terephthalate

PETG

poly(ethylene) terephthalate glycol

PFM

protein-free media

PI

propidium iodide

PI-3

parainfluenza-3 (virus)

PMS

phenazine methosulphate

PPLO

pleuropneumonia-like organisms (= mycoplasma)

PTFE

poly(tetrafluoroethylene)

PVDV

polyvinyl deoxyfluoride

QA

quality assurance (department)

QC

quality control

RFLP

restriction fragment length polymorphism

RNA

ribonucleic acid

RO

reverse osmosis

rpm

revolutions per minute

RPMI

Roswell Park Memorial Institute

RT

reverse transcriptase

SDA

Sabouraud agar

SFM

serum-free media

SGOT/AST

aspartate aminotransferase

SGPT/ALT

alanine aminotransferase

SIV

simian immunodeficiency virus

SOP

standard operating procedure

SPE

solid phase extraction

STED

stimulated emission depletion

STR

short tandem repeat (profiling)

SV40

simian virus 40

TCA

trichloroacetic acid

TIFF

tagged image file format

TIRF

total internal reflection fluorescence

TSA

tryptone soy agar

TSE

transmissible spongiform encephalopathy

TTP

thymidine triphosphate

U

units (usually of enzyme or antibiotic activity)

UDAF

unidirectional airflow (cabinet)

UKSCB

United Kingdom Stem Cell Bank

UV

ultraviolet (light)

vCJD

variant Creutzfeld–Jakob disease

WCB

working cell bank

WFI

water for injection

WHO

World Health Organization

WTS-1

2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt

WTS-3

2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, sodium salt

WTS-4

2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethyl car-bamoyl) phenyl]-2H-tetrazolium

WTS-5

2,2′-dibenzothiazolyl-5,5′-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3′-(3,3′-dimethoxy-4,4′-biphenylene)ditetrazolium, disodium salt

XTT

2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

Protocols

1 The Cell Culture Laboratory

1.1 Washing and sterilization of reusable labware

1.2 Washing glass volumetric pipettes

1.3 Environmental monitoring

2 Sterilization

2.1 Operation of a small basic benchtop autoclave

2.2 Operation of a multicycle porous load autoclave

2.3 Bowie–Dick and other tests for porous-load autoclaves

2.4 Sterilization using a hot-air oven

2.5 Fumigation of a room using formaldehyde

2.6 Fumigation of a microbiological safety cabinet

2.7 Sterilization of liquids by positive pressure membrane filtration

2.8 Use and maintenance of a microbiological safety cabinet

3 Microscopy of Living Cells

3.1 Halogen lamp alignment

3.2 Setting Ko¨hler illumination

3.3 Arc lamp alignment

3.4 Setting phase contrast

3.5 Setting up DIC

3.6 Cleaning coverslips

3.7 Time-lapse imaging

4 Basic Techniques and Media, the Maintenance of Cell Lines and Safety

4.1 Laboratory start-up (daily)

4.2 Laboratory shut-down

4.3 Subculture of substrate-adherent cells (LLC-PK1)

4.4 Preparation of attachment factor- and ECM-coated culture substrates

4.5 Haemocytometer counting for total and per cent viable cells

4.6 MTT assay

4.7 Transportation of cell cultures in LN vapour using a dry shipper

4.8 Transportation of cell cultures at ambient temperature

4.9 Reinitiation of incubation of cell cultures transported at ambient temperature

5 Development and Optimization of Serum- and Protein-free Culture Media

5.1 Preparation of media from milled powder

5.2 Sequential adaptation to a new medium

6 Cryopreservation and Banking of Cell Lines

6.1 Preparation and selection of cell cultures for cryopreservation

6.2 Harvesting cell cultures for preservation

6.3 Addition of cryoprotectant and aliquotting of cell suspensions

6.4 Cryopreservation of aliquotted cells in vials

6.5 Recovery of cells from frozen storage, and quality control

6.6 Vitrification of hES cells

7 Primary Culture of Specific Cell Types and the Establishment of Cell Lines

7.1 Isolation and growth of adipocytes

7.2 Isolation and growth of chondrocytes

7.3 Isolation and growth of dermal fibroblasts

7.4 Isolation and growth of osteoblasts

7.5 Isolation and growth of human umbilical vein endothelial cells (HUVECs)

7.6 Isolation and growth of keratinocytes on a feeder layer

7.7 Isolation and growth of keratinocytes in serum-free medium

7.8 Isolation and growth of hepatocytes

8 Cloning

8.1 Determination of colony-forming efficiency

8.2 Cloning by limiting dilution

8.3 Cloning of cells by separation within microspots in 96-well plates

8.4 Cloning by micro-manipulation

8.5 Cloning of attached cells using cloning rings

8.6 Cloning on hydrophilic FEP

8.7 Cloning in soft agar

8.8 Cloning in methylcellulose

9 The Quality Control of Animal Cell Lines and the Prevention, Detection and Cure of Contamination

9.1 Detection of bacteria and fungi by cultivation

9.2 Testing for mycoplasma by indirect DNA staining (Hoechst 33258 stain)

9.3 Detection of mycoplasma by culture

9.4 Detection of retroviruses in cell supernatants by reverse transcriptase assay

9.5 Eradication of microbial contamination by antibiotic treatment

10 Systems for Cell Culture Scale-up

10.1 Cultivation of HEK293 cells in roller bottles

10.2 Strategy for successful utilization of microcarriers

10.3 Establishment of microcarrier cultures

10.4 Roller bottle culture of a suspension cell line (Sp2/0-Ag14)

10.5 Cultivation of Sf9 cells in Erlenmeyer culture

10.6 Batch cultivation in a 2-l working volume stirred-tank bioreactor

10.7 Cultivation of mammalian cells in the Appliflex bioreactor

1

The Cell Culture Laboratory

Sue Clarke1 and Janette Dillon2

1ImmunoBiology Limited, Babraham Research Campus, Babraham, Cambridge, UK

2Medlmmune, Granta Park, Cambridge, UK

1.1 Introduction

Cell culture dates back to the early twentieth century (Table 1.1) by which time some progress had already been made in cryopreservation, the long-term storage of mammalian cells in a viable state.

Table 1.1 The early years of cell and tissue culture.

Late nineteenth century – Methods established for the cryopreservation of semen for the selective breeding of livestock for the farming industry1907 – Ross Harrison [1] published experiments showing frog embryo nerve fibre growth in vitro1912 – Alexis Carrel [2] cultured connective tissue cells for extended periods and showed heart muscle tissue contractility over 2–3 months1948 – Katherine Sanford et al. [3] were the first to clone – from L-cells1952 – George Gey et al. [4] established HeLa from a cervical carcinoma – the first human cell line1954 – Abercrombie and Heaysman [5] observed contact inhibition between fibroblasts – the beginnings of quantitative cell culture experimentation1955 – Harry Eagle [6] and, later, others developed defined media and described attachment factors and feeder layers1961 – Hayflick and Moorhead [7] described the finite lifespan of normal human diploid cells1962 – Buonassisi et al. [8] published methods for maintaining differentiated cells (of tumour origin)1968 – David Yaffe [9] studied the differentiation of normal myoblasts

The laboratory process of cell culture allows cells to be manipulated and investigated for a number of applications, including:

studies of cell function, for example metabolism;testing of the effects of chemical compounds on specific cell types;cell engineering to generate artificial tissues;large-scale synthesis of biologicals such as therapeutic proteins and viruses.

The pioneering work of Ross Harrison in 1907 [1] demonstrated that culturing tissue in vitro (in glass) not only kept cells alive, but enabled them to grow as they would in vivo (in life). However, the early development of cell culture technology was hindered by issues of microbial contamination. The growth rate of animal cells is relatively slow compared with that of bacteria. Whereas bacteria can double every 30 minutes or so, animal cells require around 24 hours. This makes animal cell cultures vulnerable to contamination, as a small number of bacteria soon outgrow a larger population of animal cells. However, tissue culture became established as a routine laboratory method by the 1950s with the advent of defined culture media devised by Eagle and others. The discovery of antibiotics by Fleming was of course another major milestone that facilitated prolonged cell culture by reducing contamination issues.

In the 1940s and 1950s major epidemics of (among others) polio, malaria, typhus, dengue and yellow fever stimulated efforts to develop effective vaccines. It was shown in 1949 that poliovirus could be grown in cultures of human cells, and this became one of the first commercial ‘large-scale’ vaccine products of cultured mammalian cells. By the 1970s methods were being developed for the growth of specialized cell types in chemically defined media. Gordon Sato and his colleagues [10] published a series of papers on the requirements of different cell types for attachment factors such as high molecular weight glycoproteins, and hormones such as the insulin-like growth factors. These early formulations and mixtures of supplements still form the basis of many basal and serum-free media used today (see Chapters 4 and 5).