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 ............................................................................................................