Animal Cell Culture: Principles and Practice

332 Pages·2023·11.027 MB·English

by  Shalini Mani, Manisha Singh, Anil Kumar

Techniques in Life Science and Biomedicine for the Non-Expert
Series Editor: Alexander E. Kalyuzhny
Shalini Mani
Manisha Singh
Anil Kumar
Animal Cell
Culture:
Principles
and Practice
Techniques in Life Science and Biomedicine
for the Non-Expert
Series Editor
Alexander E. Kalyuzhny, University of Minnesota
Minneapolis, MN, USA
The goal of this series is to provide concise but thorough introductory guides to
various scientific techniques, aimed at both the non-expert researcher and novice
scientist. Each book will highlight the advantages and limitations of the technique
being covered, identify the experiments to which the technique is best suited, and
include numerous figures to help better illustrate and explain the technique to the
reader. Currently, there is an abundance of books and journals offering various
scientific techniques to experts, but these resources, written in technical scientific
jargon, can be difficult for the non-expert, whether an experienced scientist from a
different discipline or a new researcher, to understand and follow. These techniques,
however, may in fact be quite useful to the non-expert due to the interdisciplinary
nature of numerous disciplines, and the lack of sufficient comprehensible guides to
such techniques can and does slow down research and lead to employing inadequate
techniques, resulting in inaccurate data. This series sets out to fill the gap in this
much needed scientific resource.
Shalini Mani • Manisha Singh • Anil Kumar
Animal Cell Culture:
Principles and Practice
Shalini Mani Manisha Singh
Centre for Emerging Diseases, Department Centre for Emerging Diseases, Department
of Biotechnology of Biotechnology
Jaypee Institute of Information Technology Jaypee Institute of Information Technology
Noida, Uttar Pradesh, India Noida, Uttar Pradesh, India
Anil Kumar
Director, Education
Rani Lakshmi Bai Central Agricultural
University
Jhansi, Uttar Pradesh, India
ISSN 2367-1114 ISSN 2367-1122 (electronic)
Techniques in Life Science and Biomedicine for the Non-Expert
ISBN 978-3-031-19484-9 ISBN 978-3-031-19485-6 (eBook)
https://doi.org/10.1007/978-3-031-19485-6
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Preface
The cell culture techniques serve as an imperative and very useful model system in
current biological research and are an important tool in understanding varied cellu-
lar physiological processes along with screening of toxic/therapeutic compounds
for various disorders. This research domain has successfully progressed signifi-
cantly over the years and proved to be used in a multitude of areas like – drug/vac-
cine production, functional enzyme, growth factors, toxicity analysis and many
more. Equally, the research community has witnessed a lot of advancements and
up-gradation through the inclusion of new assays, concepts, qualitative and quanti-
tative techniques, etc., which has added enormous value in this ever-growing area of
life sciences. They have proved to be a beneficial tool for healthcare scientists and
provide a model system for physicochemical evaluation of selected cells to be stud-
ied. Their analysis and behaviour help in determining the exact pathway along with
their metabolic rate, and the interaction with drugs proves to be a useful tool for
drug screening programmes, clinical trials, and pharmaceutical designing. The in
vitro cell culture systems help in exploring the principles of Ayur-biology and nutri-
tional biology through development biomarker(s)–based bioassays. The knowledge
derived from such experimentation helps not only for screening of herbal medi-
cines, drugs, nutraceuticals, pharmaceuticals and cosmeceuticals but also to under-
stand the mode of action and other information needed to evaluate their efficacy.
Early cell culture research techniques were mainly focused on discovering meth-
ods for culturing a diverse array of cells from many species, but today, cell culture
methods have added new dimensions to the science research and its authenticity.
And with an increasing count of assays, analysis techniques and advanced instru-
mentation added every now and then to gain in-depth basic knowledge of this area
are an utmost requirement now, and we believe that there are many who can benefit
from the combined knowledge and experience of basics of cell culture and its appli-
cation. With this aim, this book provides the in-depth crisp and relevant knowledge
on the topic right from distinguishing the cell lines, laboratory design and safety to
aspects of cryopreservation, quality control and cell line authentication, to students
and researchers new to cell culture handling. The issue of cell line misidentification
and cross contamination has been recognised as a significant problem in recent
v
vi Preface
years, and solutions are addressed here. Additionally, a series of detailed protocols
is also provided that is routinely used in the cell culture laboratories. Therefore, this
book is intended as an in-depth text of cell culture with end-to-end answers to all the
queries for beginners as well as the broader research community. This comprehen-
sive research-based guide provides the kind of intensive description and implemen-
tation advice that is crucial for getting optimal results in the laboratory.
This book also provides the updated and latest cell culture techniques with more
accepted methods of experiments. The well-researched and detailed protocols, men-
tioned in the different chapters of the book, can be readily used in laboratory prac-
tices by the researchers and students. The volume begins with a detailed and
informative introduction chapter that includes the basic background of cell culture
techniques and further explains standard layout of a cell culture lab, good lab prac-
tices in cell culture lab, maintaining sterility in cell culture lab, media preparation,
and selection of suitable media. After explaining the basic techniques used in cell
culture such as cell counting, toxicity estimation, passaging, maintenance and freez-
ing of cell lines, the book is dedicated to discussing few advanced techniques in cell
culture lab such as stem cell culture and 3D cell culture too. The importance and
application of these techniques in varied research fields, and a plethora of good
practical advice along with the trouble shooting for the expected technical glitches
while performing the experimentation are also discussed. Later in the book, we have
discussed the application of the various assays or analytical tools discussed in the
previous chapters to the readers with the wholesome view and concept development
of the related topic. As animal cell culture is mostly represented as experimental
model systems in basic and medical sciences, so we have discussed its application
in some selected areas of study such as pathological studies, biotechnology, cellular
development and differentiation, biomarker identification, and genetic manipula-
tion. We have also discussed the ethical perspectives and the guidelines that should
be followed while practising the cell culture techniques in these areas of research.
Thus, the book summarises the broad information on mainly basic, fundamental and
specialised knowledge which includes historical perspective, utilisation, R&D
efforts, present status and the importance being given by policymakers for use of
animal cell culture techniques, tools for development of superior biologically
important products like vaccine, interferon, monoclonal antibodies even now avail-
ability of stem cells for not only diagnostics but also for prophylactic, preventive
and therapeutic purposes for healthy society. The book is written keeping in view
syllabi of different research institutions, researchers and students as well require-
ment of the industry. It will serve as instructional material for researchers in bio-
medical science, veterinary science, process engineering, biochemistry and
biotechnology, and reference material for those working in industry and R&D labs.
Noida, Uttar Pradesh, India Shalini Mani
Noida, Uttar Pradesh, India Manisha Singh
Jhansi, Uttar Pradesh, India Anil Kumar
Contents
1 Overview to Animal Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Shalini Mani
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Types of Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1 Primary Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.2 Secondary Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.3 C ell Line and Cell Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3 Ethical Considerations in Animal Tissue Culture . . . . . . . . . . . . . . 6
1.4 Common Nomenclatures in Animal Cell Culture . . . . . . . . . . . . . . 7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2 Cell Culture Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Anvi Jain, Aaru Gulati, Khushi R. Mittal, and Shalini Mani
2.1 Laboratory Design, Planning and Layout . . . . . . . . . . . . . . . . . . . . 11
2.1.1 Ventilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.2 Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.3 Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.4 Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2 Equipment and Materials for Cell Culture Laboratory . . . . . . . . . . 18
2.2.1 Aseptic Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.2.2 Incubation and Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.2.3 Preparation and Sterilisation . . . . . . . . . . . . . . . . . . . . . . . . 36
3 Good Laboratory Practices in Animal Cell Culture Laboratory
and Biosafety Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Kumari Yukta, Mansi Agarwal, Mekhla Pandey, Khushi Mittal,
Vidushi Srivastava, and Shalini Mani
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.2 Biosafety Measures in Animal Cell Culture Laboratory . . . . . . . . . 55
3.2.1 Biosafety Level 1 (BSL-1) . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.2.2 G LPs for Biosafety Level 1 . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.2.3 Biosafety Level 2 (BSL-2) . . . . . . . . . . . . . . . . . . . . . . . . . . 57
vii
viii Contents
3.2.4 G LPs for Biosafety Level 2 . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.2.5 Biosafety Level 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.2.6 G LPs for Biosafety Level 3 . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.2.7 Biosafety Level 4 (BSL-4) . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.2.8 G LPs for Biosafety Level 4 . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.2.9 Safe Laboratory Practices . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.2.10 C ommon Good Lab Practices (GLPs) in Cell Culture. . . . . 59
3.2.11 Waste Segregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.2.12 Types of Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.2.13 Waste Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4 Managing Sterility in Animal Cell Culture Laboratory . . . . . . . . . . . 65
Shalini Mani
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.2 Elements of Aseptic Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.2.1 Sterile Work Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.2.2 Good Personal Hygiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.2.3 Media and Sterile Reagents . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.2.4 Sterile Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.2.5 Use of Safety Cabinets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.2.6 Culture Sterility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.3 Biological Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.3.1 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.3.2 Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.3.3 Moulds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.3.4 Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.3.5 Mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.3.6 Cross-contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.4 Aseptic Technique Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
5 Media and Buffer Preparation for Cell Culture . . . . . . . . . . . . . . . . . 77
Sakshi Tyagi and Shalini Mani
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.2 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.2.1 Physiochemical Properties of Media . . . . . . . . . . . . . . . . . . 78
5.2.2 Components of Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
5.2.3 Types of Culture Medium . . . . . . . . . . . . . . . . . . . . . . . . . . 82
5.3 Methodology to Prepare Cell Culture Media . . . . . . . . . . . . . . . . . . 84
5.3.1 P reparation of Incomplete DMEM Media . . . . . . . . . . . . . . 84
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6 Properties of Cultured Cells and Selection of Culture Media . . . . . . 89
Shalini Mani
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
6.2 Systems for Growing Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Contents ix
6.3 Morphological Differences in Mammalian Cell . . . . . . . . . . . . . . . 90
6.4 Maintaining Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
6.5 When to Subculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
6.5.1 Cell Density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.5.2 Exhaustion of Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.5.3 Subculture Schedule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.6 Media Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.6.1 Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.6.2 Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.6.3 pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
6.6.4 CO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
2
6.6.5 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
6.6.6 D ifferent Culture Mediums for Different Cells . . . . . . . . . . 96
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
7 Selection and Maintenance of Cultured Cells . . . . . . . . . . . . . . . . . . . 99
Divya Jindal and Manisha Singh
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
7.2 Origin and Characterisation of Cells . . . . . . . . . . . . . . . . . . . . . . . . 100
7.2.1 Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
7.3 Selection of Appropriate Cell Line . . . . . . . . . . . . . . . . . . . . . . . . . 101
7.3.1 T echniques for Detachment of Cells . . . . . . . . . . . . . . . . . . 104
7.3.2 Source of Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
7.3.3 Subculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
7.3.4 Growth Conditions and Characteristics . . . . . . . . . . . . . . . . 107
7.3.5 Other Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
7.4 Maintenance of Cell Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
7.5 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
7.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
8 Inoculation and Passaging of Adherent and Suspension Cells . . . . . . 115
Pranav Pancham, Divya Jindal, and Manisha Singh
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
8.2 Subculturing in Monolayer Cultures . . . . . . . . . . . . . . . . . . . . . . . . 116
8.3 Troubleshooting Monolayer Cell Subculturing . . . . . . . . . . . . . . . . 117
8.3.1 Dissociation of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
8.3.2 Cell Clump Formation Post-dissociation . . . . . . . . . . . . . . . 118
8.3.3 Cells Reattachment Issues . . . . . . . . . . . . . . . . . . . . . . . . . . 118
8.3.4 Reduced Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
8.4 Subculturing in Suspension Cell Cultures . . . . . . . . . . . . . . . . . . . . 119
8.5 Media Consumption by Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . 119
8.6 Types of Cell Detachment Techniques . . . . . . . . . . . . . . . . . . . . . . . 120
8.6.1 Degree of Cell Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
8.6.2 Use of Detached Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
8.6.3 Process Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121