Contents
Introduction 1
Purpose of the Handbook 1
Introduction to Cell Culture . 2
What is Cell Culture? . 2
Finite vs Continuous Cell Line 2
Culture Conditions 2
Cryopreservation . 2
Morphology of Cells in Culture . 3
Applications of Cell Culture 3
Cell Culture Laboratory 4
Safety . 4
Biosafety Levels 4
SDS 5
Safety Equipment 5
Personal Protective Equipment (PPE) . 5
Safe Laboratory Practices 5
Cell Culture Equipment . 6
Basic Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Expanded Equipment . 6
Additional Supplies . 6
Cell Culture Laboratory 7
Aseptic Work Area 7
Cell Culture Hood . 7
Cell Culture Hood Layout 8
Incubator 9
Storage 9
Cryogenic Storage . 10
Cell Counter . 10

Aseptic Technique 11
Introduction . 11
Sterile Work Area 11
Good Personal Hygiene . 11
Sterile Reagents and Media 12
Sterile Handling 12
Aseptic Technique Checklist 13
Biological Contamination 14
Introduction . 14
Bacteria . 14
Yeasts . 15
Molds . 15
Viruses 16
Mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Cross-Contamination 17
Using Antibiotics 17
Cell Culture Basics . 18
Cell Lines . 18
Selecting the Appropriate Cell Line . 18
Acquiring Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Culture Environment . 19
Adherent vs Suspension Culture 19
Media . 20
pH . 21
CO2 21
Temperature 21
Cell Morphology . 22
Mammalian Cells 22
Variations in Mammalian Cell Morphology 22
Morphology of 293 Cells . 23
Insect Cells . 24
Morphology of Sf21 Cells 24
Morphology of Sf9 Cells 25

Methods . 26
Guidelines for Maintaining Cultured Cells 26
What is Subculture? 26
When to Subculture? 27
Media Recommendations for Common Cell Lines . 28
Dissociating Adherent Cells . 30
TrypLE™ Dissociation Enzymes 30
Subculturing Adherent Cells . 31
Materials Needed . 31
Protocol for Passaging Adherent Cells 31
Notes on Subculturing Adherent Insect Cells 32
Subculturing Suspension Cells . 33
Passaging Suspension Cultures 33
Suspension Culture Vessels 33
Materials Needed . 34
Protocol for Passaging Suspension Cells . 34
Notes on Subculturing Suspension Insect Cells 36
Freezing Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Cryopreservation . 37
Guidelines for Cryopreservation . 37
Freezing Medium . 38
Materials Needed . 38
Protocol for Cryopreserving Cultured Cells 39
Thawing Frozen Cells . 40
Guidelines for Thawing . 40
Materials Needed . 40
Protocol for Thawing Frozen Cells 40
Support Protocols 41
Counting Cells in a Hemacytometer . 41
Trypan Blue Exclusion 42
Concentrating Cells . 42

Appendix 43
Troubleshooting . 43
Cell Culture Products . 44
Cell Lines . 44
Media for Mammalian Cell Culture 45
Media for Insect Cell Culture 46
Serum Products for Cell Culture 46
Laboratory Reagents for Cell Culture 47
Antibiotics and Antimycotics . 48
Accessory Products for Cell Culture . 49
Growth Factors and Purified Proteins . 49
Transfection and Selection . 50
Transfection Reagents 51
Additional Resources 52
Mammalian and Insect Cell Cultures . 52
Cell and Tissue Analysis . 52
Transfection Selection Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Safety Data Sheets . 52
Certificate of Analysis . 52
Technical Support 53

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