Dr. M Ravi Ph.D. MNASc
Dr. M Ravi obtained his doctorate in Human Genetics from Sri Ramachandra University. He is presently an Associate Professor in the Department of Human Genetics of Sri Ramachandra University, Chennai and fields of interest and specializations are Immunology, Immunotechniques, Animal Cell Cultures and Research Methodology. An author of several research papers and a book, he is also a Member of The National Academy of Sciences, India and The Editor of Advanced Biotech, a monthly journal of Biotechnology.
E mail: maddalyravi@hotmail.com
Prof. Solomon F.D Paul Ph.D.
Dr. Solomon F.D. Paul obtained his doctorate from Madras University for research carried out on Biodosimetry at IGCAR, Kalpakkam. He is the current Professor and Head of the Department, Departments of Human Genetics and Biomedical Sciences and also the Principal, Faculty of Biomedical Sciences, Technology and Research, Sri Ramachandra University. His research interests and expertise are Biodosimetry, Human Genetic Diagnosis and Genotoxicity studies. An author of several research papers and a book, he has guided many PhD scholars and continues to be a research guide and advisor for many research programmes.
E mail: wise_soly@yahoo.com
Dr. V Ganesh Ph.D.
Dr V Ganesh obtained his Doctorate from Madras University for research carried out in Radioimmunoscintigraphy at Cancer Institute, Chennai. During his post doctoral tenure in USA, he worked on antibody engineering for radioimmunotherapy of colon carcinomas & combination chemotherapy targeted to prostate cancers. His research interests and expertise are in the field of Cancer Biology, Nanobiology and Targeted drug delivery. Dr Ganesh has published his research work in high impact factor journals, is the Principle Investigator for several funded projects and a PhD guide.
Email: gvenkat16@gmail.com
Preface III
Contents IV
Abbreviations XI
List of Figures/Plates XIII
Section A - Essential Concepts in Cell Culture
Chapter 1 1
Essential Concepts in Cell Culture
A.1.1. Introduction
A.1.2. Overview of the Mammalian Cell Cycle
A.1.3. Mitotic proteins and Mitotic Factors
A.1.4. Phases of Cell Growth in vitro
A.1.5. Notes
Chapter 2 5
Types of Cell Cultures
A.2.1. Introduction
A.2.2. Established Cell Cultures
A.2.3. Primary Cell Cultures
A.2.4. Explant Cultures
A.2.5. Organotypic Cell Cultures
Chapter 3 8
Media, Buffers and Other Consumables
A.3.1. Introduction
A.3.2. Culture Medium
A.3.3. Supplements
A.3.4. Buffers
A.3.5. Glass and Plastic ware
Chapter 4 14
Contaminations in Cell Cultures
A.4.1. Introduction
A.4.2. Types of Contamination
A.4.3. Sources of Contamination
A.4.4. Conclusion
Section B - Culturing Cells 18
Chapter 5 20
Monolayer/Attached Cell Cultures
B.2.1. Introduction
B.2.2. Trypsinization
B.2.3. Cell Scrapers
B.2.4.Interpretation of Results
B.2.5. Notes
Chapter 6 23
Suspension Cell Cultures
B.3.1. Introduction
B.3.2. Materials and Methods
B.3.3. Interpretation of Results
B.3.4. Notes
Chapter 7 25
Cell Cycle Synchronization and Harvest
B.4.1. Introduction
B.4.2. Materials and Methods
B.4.3. Notes
Chapter 8 30
Cell Cycle Analysis By Flowcytometry
B.5.1. Introduction
B.5.2. Materials & Methods
B.5.3. Interpretation of Results
B.5.4. Notes
Chapter 9 33
Conditioned Medium
B.6.1. Introduction
B.6.2. Materials and Methods
B.6.3. Notes
Chapter 10 36
Elispot / Cell Elisa
B.7.1. Introduction
B.7.2. Materials and Methods
B.7.3. Interpretation of Results
B.7.4. Notes
Chapter 11 39
Ennumerating Cells and Viability Assays
B.8.1. Introduction
B.8.2. Enumerating Cells
B.8.3. Trypan Blue Assay
B.8.4. Interpretation of Results
B.8.5. Notes
Chapter 12 44
Assays for Apoptosis & Necrosis
B. 9. 1. Introduction
B. 9. 2. Materials and Methods
B. 9. 3. Interpretation of Results
B. 9. 4. Notes
Chapter 13 49
Proliferation Assay for Cell Cultures
B.10.1. Introduction
B.10.2. Clonogenic Assay
B.10.3. MTT Assay
B.10.4. Interpretation of Results
B.10.5. Notes
Chapter 14 54
Cell Migration Assay
B. 11.1. Introduction
B. 11. 2. Materials and Methods
B. 11. 3. Interpretation of Results
B. 11. 4. Notes
Chapter 15 59
Fluorescent Actin Staining of Adherent Cells
B. 12. 1. Introduction
B. 12. 2. Materials and Methods:
B. 12. 3. Interpretation of Results
B. 12. 4. Notes:
Chapter 16 62
Establishing Cells as 3-D Aggregates
B.13.1. Introduction
B.13.2. Preparation of the Agarose Hydrogel
B.13.3. Seeding of Cells onto the Agarose Hydrogel
B. 13.4. Interpretation of Results
B. 13.5. Notes
Chapter 17 67
Cryopreservation and Revival of Cell Lines
B.14.1. Introduction
B.14.2. Cryopreservation
B.14.3. Interpretation of Results
B.14.4. Notes
Chapter 18 71
Hybridoma Technology
B.15.1. Introduction
B.15.2. Immunizations and Collection of Primed
B.15.3. Notes
B.15.4. Somatic Cell Hybridization for Hybridoma Generation
B.15.5. Limiting Dilution And Hat Selection
B.15.6. Screening Techniques for Hybridoma Generation
B.15.7. Expansion of Positive Hybridoma Clones
B.15.8. Maintenance and Passaging of Hybridoma Cultures
B.15.9. Harvesting Mabs From Culture Supernatants
B.15.10. Preservation of Positive Clones and Reculture
Chapter 19 98
Cell Co-Cultures
Chapter 20
Generation of Human Tumor Xenograft – Murine Models
B.17.1. Introduction 100
B.17.2. Materials and Methods
B.17.3. Interpretation of Results
B.17.4. Notes
Chapter 21 104
Cardiomyocyte Cultures–Avian Model
B.18.1. Introduction
B.18.2. Initiation of in vitro Culture
B.18.3. Enrichment of Cardiomyocytes
B.18.4. HH Stage 18 Chick Embryo Cardiomyocyte Cell Culture
B.18.5. HH Stage 34 Chick Embryo Cardiomyocyte Cell Culture
B.18.6. HH Stage 18 & 34 Chick Embryo Organ Explant Cardiomyocyte Culture
B.18.7. Potential application areas of in vitro Cardiomyocyte models 121
Section C - Essential Requirements 122
Chapter 22 124
Infrastructure for Cell-Culture Facility
Introduction
Cell Culture Lab Design – General Considerations
Laminar Flow
Incubators
Microscopes
Centrifuges
Other Considerations
Conclusion
Chapter 23 132
Standard Operating Procedures (SOPS)
And Good Laboratory Practices (GLPS)
Introduction
Dress Code
Preparations Before Entering The Lab
Inside the Lab
Laminar Flow Cabinet
Incubator
Water Bath
Pipettes
Medium/Serum/Buffer Bottles, etc.
Culture Flasks
Culture-plates & Petri-plates
Cryopreservation
Refrigerator
Other Considerations
Chapter 24 142
Sources of Cell Lines
C.4.1. Introduction
C.4.2. Cell Line Repositories 152
Section D - Model Cell Cultures – Fact Sheet 153
Chapter 25 154
Vero Cells
Chapter 26 155
Chinese Hamster Ovary (cho) Cells
Chapter 27 156
Hela Cells
Chapter 28 157
Sp2/0-ag14 Cells
Chapter 29 158
U266
Chapter 30 159
Oaw-42
Chapter 31 160
HeK 293
Chapter 32 161
HepG2
Chapter 33 162
WI-38
Chapter 34 163
MCF 7
Chapter 35 164
PC 12
Chapter 36 165
HEP 2
Chapter 37 166
A549 CELLS
Chapter 38 167
Neuro2a Cells
Chapter 39 168
H460 CELLS
Chapter 40 169
Pa1 CELLS 43
Chapter 41
NCI-H23 170
Annexure 1 - Buffers/Reagents For Cell Culture 171
Annexure 2 - Glossary of Terms 183
Annexure 3 - Plates 204
Index 216
PREFACE
Cell Cultures are used for a variety of purposes. One of the first documented works towards this is by Sydney Ringer, an English physiologist who attempted to retain the function of a heart ex-vivo. Several pioneering developments in understanding physiology, basic sciences and engineering lead to a phenomenal growth and advancements in cell culture techniques. From understanding complex mechanisms of functions to production of important bio-molecules, cell cultures are inevitable. Also, the direction of many studies is moving from in vivo to ex-vivo to in vitro models for obvious reasons. Cells in culture are easily manageable, can be subjected to varied experimental conditions and can be studied for various parameters which can be irrevocably documented. Also, cells in culture represent true living components which can be utilized for stimulating a 'near in-vivo' environment for studies otherwise not possible. Cell culture has become an integral part of many academic, research and industrial organizations.
Several cell lines, culture techniques, consumables, equipment and related infrastructure are available and utilized currently. The right choice of all the above can be crucial for a good thriving cell line and also for obtaining the desired net result. Looking around, we see only textbooks, references and laboratory protocol material that are quite complex, of which only a select part might be of routine use to many a student, research scholar or a laboratory technician. From an understanding of this and inputs that we give and receive from our students and research scholars, that we felt the need for a simple, yet a comprehensive book on Animal Cell Culture that deals with essential at the same time, universal applications. A strong foundation and inputs in basics should sure go a long way for any laboratory practitioner to perform more complicated and seemingly difficult techniques with ease.
Many people were instrumental in giving us the motivation and confidence to bring out this manual. We are indebted to our mentors and teachers, Dr. Seetharami Reddy,
Dr. S. Krishnaswamy, Dr. Vikram R. Jayanth, Dr. A. Meenakshi, Dr. Vinod Kumar Panikar, and Shri. A. R. Sunderarajan.
All our students and research scholars are a great source of continued inspiration.
We acknowledge the contributions of Ms. Rohini Nair and Mr. U. Baraneedhran for contributing one chapter each on the Sources of Cell Lines and Cardiomyocyte Cultures in an Avian Model respectively, which has given a value addition for this book.
We thank the management of Sri Ramachandra University for their constant motivation and encouragement which always is our source of strength to provide with the best academic and research platform to our students.
We thank Shri. K.R. Pillai and the entire team at Samanthi Publications for their valuable inputs and excellent publishing standards which was of immense help for this book to materialize. We also thank the Sujatha-Suraj duo for their help in editing the manuscript.
We sincerely hope this book on Animal Cell Culture will be of use to all involved in this exciting area of life sciences.
Dr. M Ravi Ph.D., MNASc
Prof. Solomon F.D Paul Ph.D.
Dr. V Ganesh Ph.D.
List of Plates and Figures
Plates
Plate 1 – Cell Morphologies
Plate 2 – Monolayers
Plate 3 – Cell Culture Stages
Plate 4 – Clonogenic Assay
Plate 5 – Cell Migration Assay
Plate 6 – 2D vs 3D
Plate 7 – Hybridoma Clone Development
Plate 8 – Non Enriched Atrial Cardiomyocyte Culture
Plate 9 – Non-enriched Ventricular Cardiomyocyte Culture
Plate 10 – Enriched Ventricular Cardiomyocyte Culture
Plate 11 – Organ Cardiomyocyte Culture
Plate 12 – Mitotic Cell Harvest
Plate 13 – Feeder Cell Preparation
Plate 14 – Thawing Cell Lines
Plate 15 – Cryopreservation
Figures:
Figure 1 – Enumerating Cells
Figure 2 – Actin Staining
Figure 3 – Hybridoma Clones
Figure 4 – Human Tumor Xenografts