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An Introduction to Cell Culture

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An Introduction to Cell Culture An Introduction to Cell Culture PSR Introduction This handbook is intended as an introduction to cell culture basics, covering topics such as getting familiar with the requirements of a laboratory dedicated to cell culture experiments, laboratory safety, aseptic technique, and microbial contamination of cell cultures, as well as providing basic methods for passaging, freezing, and thawing cultured cells. Golchin. A 12/30/2015 Contents Introduction to Cell Culture .......................................................................................................... 19 What is Cell Culture? ................................................................................................................ 19 Classification of Cell Cultures .................................................................................................. 19 Primary Culture ..................................................................................................................... 19 Secondary Culture ................................................................................................................. 19 Cell Line .................................................................................................................................... 19 Types of cell culture .................................................................................................................. 20 Record Keeping ............................................................................................................................ 20 Morphology of Cells in Culture ................................................................................................ 21 Contact Inhibition ...................................................................................................................... 22 Culture Conditions .................................................................................................................... 22 Cell Culture Laboratory ................................................................................................................ 22 Safety Aspects of Cell Culture .................................................................................................. 22 Risk Assessment .................................................................................................................... 22 Operational standards.................................................................................................................... 24 Training ..................................................................................................................................... 24 Access Control and Biosecurity ................................................................................................ 24 PPE ................................................................................................................................................ 24 Shoes ......................................................................................................................................... 24 Lab coats ................................................................................................................................... 24 Face Protection .......................................................................................................................... 24 Gloves........................................................................................................................................ 25 Biohazards ................................................................................................................................. 25 Safety Equipment ...................................................................................................................... 25 Disinfection ............................................................................................................................... 26 Waste Disposal .......................................................................................................................... 26 Cell Culture Equipment ................................................................................................................ 27 Basic Equipment ....................................................................................................................... 27 Expanded Equipment ................................................................................................................ 27 Additional Supplies ................................................................................................................... 27 16 Research Center for Pharmaceutical Nanotechnology, University Avenue,Tabriz, Iran URL: nano.tbzmed.ac.ir Email: [email protected] Aseptic Work Area ....................................................................................................................... 28 Materials .................................................................................................................................... 28 Cell Culture Hood ......................................................................................................................... 29 Class I ........................................................................................................................................ 29 Class II....................................................................................................................................... 29 Class II....................................................................................................................................... 29 Class III ..................................................................................................................................... 29 Use of the horizontal laminar-flow clean bench ........................................................................... 29 General principles of handling cell cultures ................................................................................. 30 Sterile Liquid Transfers ............................................................................................................. 31 Working in hoods ...................................................................................................................... 32 Preparation of cell growth medium............................................................................................... 34 Basic Constituents of Media ..................................................................................................... 35 Inorganic Salts ....................................................................................................................... 35 Buffering Systems ................................................................................................................. 35 Carbohydrates ........................................................................................................................ 36 Amino Acids .......................................................................................................................... 36 Vitamins................................................................................................................................. 36 Proteins and Peptides ............................................................................................................. 36 Fatty Acids and Lipids ........................................................................................................... 36 Trace Elements ...................................................................................................................... 36 Freezing and Thawing Cells ......................................................................................................... 36 Adherent Cells ........................................................................................................................... 37 Freeze Procedure ................................................................................................................... 37 Thaw Procedure......................................................................................................................... 37 Suspension Cells ....................................................................................................................... 37 Freeze Procedure ................................................................................................................... 38 Thaw Procedure......................................................................................................................... 38 Key Points ................................................................................................................................. 38 Water ............................................................................................................................................. 39 Incubators ...................................................................................................................................... 39 17 Research Center for Pharmaceutical Nanotechnology, University Avenue,Tabriz, Iran URL: nano.tbzmed.ac.ir Email: [email protected] Centrifuges .................................................................................................................................... 40 Storage area ................................................................................................................................... 40 Biological Contamination ............................................................................................................
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