In the name of God
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Cell culture
What is cell culture?
Cultivation of organs, tissues and cells of organisms in a laboratory environment
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Why do we culture cells?
The study of cells in terms of how they grow, their nutritional needs and the reasons for their stop growth
The study of evolution
Genetic studies, including the production of transgenic animals that can express certain genes
Production of monoclonal antibodies
Toxicological tests
Vaccination against viral diseases
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Cell culture laboratory
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Cell culture laboratory equipment
CO2 Incubators
Laminar flow hood
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Cell culture laboratory equipment
Microscope
Centrifuge
Water bath
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Cell culture laboratory equipment
Vacuum pump
Autoclave
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Cell culture laboratory equipment
Controlled-Rate Freezer
Refrigerator and Freezer
Cryogenic Storage Tanks
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Cell culture consumables
Cell culture plate
Cell culture flask
Cell Scraper
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Cell culture consumables
pipette
Pipette tips
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Cell culture consumables
Centrifuge Tubes
Cryotube
Cell Strainer
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Types of cell culture
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Organ culture
Primary culture or Explanation
Continuous cultures
Types of cell culture model
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Morphological characteristics of cells in culture
Adherent cells Suspend cells
(Semi-adherent)
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Types of cell morphology
Fibroblast
Epithelial
Lymphoblast- like
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Growth curve and cell growth phases
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Cell culture environment and its characteristics
Composition
Liquid
Serum (5-20%)
Antibiotics (1%)
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Cell culture environment and its characteristics
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Buffer system:
Necessary Ph for survival of the cells: 7.2-74
Exception: Fibroblast cells: 7.4-7.7
Natural buffer system: Dissolution of carbon dioxide gas in the cultivation environment and creating a chemical balance between carbonate and bicarbonate ions
Chemical buffer system: By zwitterionic compounds such as Hepes (N-2-hydroxy-ethylpiperazine-N'-2-ethenesulfanic acid) and Mops (Morpholinopropane sulfonic acid)
Cell culture environment and its characteristics
Phenol red in culture medium is the indicator of pH
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Cell culture stages
Thawing
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Cell culture stages
Passaging Adherent cell
Suspend cell
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Cell culture stages
Cell counting and culture for test
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Cell culture stages
Freezing
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Contamination
Microbial contamination
Inter- and intra-species cross-contamination Parasites
Prions
Chemical, biological, and other nonliving contaminants
Contamination
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Microbial contamination
The most important cause of the contamination is inappropriate laboratory conditions such as the use of old buildings with inadequate ventilation system and use of inexperienced personnel in the stages of work, especially the aseptic stages.
Daily assay of flasks and other daily controls of the culture room will easily detect contaminations. It should be noted that any kind of process and handling of cells and consumables can cause contamination.
Microbial contamination Virus
Bacteria Fungi
Mycoplasma
Parasites Prions
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Microbial contamination
bacteria
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Microbial contamination
Fungi (Yeast and mold)
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Microbial contamination
Mycoplasma
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Microbial contamination
Prion
Prions are devoid of nucleic acids and consist primarily of protein termed PrPSc.
Although most cell lines are resistant to prion infection, some cells lines are susceptible to prions and can promote stable and persistent replication of prions.
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They
can be included in cell culture media
enriched with serum of bovine origin.
Prions are difficult to inactivate.
Microbial contamination
Parasites
Different intracellular protozoan parasites (e.g., Toxoplasma gondii, Trypanosoma cruzi, Leishmania spp.) may be included in freshly prepared primary cell cultures originating from a donor organism that is known or suspected to be infected with respective parasites.
Special safety precautions should be considered and protective clothing and equipment
might be necessary.
Needles and other sharp objects should be omitted when working with parasite-infected cell lines.
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Microbial contamination
Virus
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Leland DS, Ginocchio CC. Role of cell culture for virus detection in the age of technology. Clinical microbiology reviews. 2007 Jan;20(1):49-78.
Inter- and intra-species cross-contamination
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Chemical, biological, and other nonliving contaminants
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Endotoxin/lipopolysaccharides, detergents, radicals, hormones, growth
factors, metals, residues of disinfectants and cleaning agents, plasticizers, and other
impurities can impact proper cell growth.
Chemical contamination can result from contaminated reagents, water, sera or some culture additives. In addition, detergents or other deposits on storage vessels, glassware, pipettes or instruments introduced by disinfection can be sources of contamination.
Plastic tubing and storage bottles can release plasticizers. Free radicals can be generated by photoactivation of tryptophan, riboflavin, or buffering agents (e.g., HEPES and PIPES) when exposed to extensive visible or fluorescent light.