Cell Culture
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63 Terms
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Tissue culture
\-Removal of cells, tissues or organs from a plant or animal into an environment for growth
\-similar biochemically and physiologically to parent tissues
\-used to prepare finite or continuous tissues
\-similar biochemically and physiologically to parent tissues
\-used to prepare finite or continuous tissues
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Organ culture
\-culture of whole organs or organ fragments to continue function
\-used for metabolism and drug studies
\-mimics what happens in vitro
\-used for metabolism and drug studies
\-mimics what happens in vitro
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Cell culture
\-cells are removed from the organ fragments disrupting normal relationships with neighboring cells
\-in vitro experiments
\-in vitro experiments
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Mammalian cell culture
\-cell culture of mammalian cells
\-much more difficult to culture
\-demand complex media
\-no way to fight off microbes
\-much more difficult to culture
\-demand complex media
\-no way to fight off microbes
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Media
Liquid that cells are growing in
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Why is cell culture important?
1. Research - overcome problems in studying cellular behavior and reduce animal use
2. Commercial or large scale production (vaccines, antibodies, etc)
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What are the two types of cell cultures?
1. Primary culture
2. Continuous culture
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Primary culture
\-derived directly from excised tissue
\-morphologically similar to the parent tissue
\-senescence
\-morphologically similar to the parent tissue
\-senescence
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Trypsin
Used to get rid of proteins that stick cells together
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Senescence
Limited number of cell division
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Advantages of primary cell cultures
\-represent the best experimental models for in vivo situation
\-have the same karyotype as the parent tissue
\-differentiated (can create different types of cells)
\-have the same karyotype as the parent tissue
\-differentiated (can create different types of cells)
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Disadvantages of primary cell culture
\-difficult to obtain
\-relatively short life span
\-very susceptible to contamination
\-might not act like tissue (artificial environment)
\-relatively short life span
\-very susceptible to contamination
\-might not act like tissue (artificial environment)
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Tumor primary cell culture
\-easier to create since they never reach senescence
\-often produce own growth factors
\-often produce own growth factors
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Finite cell cultures (secondary or subclone culture)
\-finite cell cultures formed after first subculture
\-senesce (limited number of cell divisions)
\-more stable than primary cells
\-senesce (limited number of cell divisions)
\-more stable than primary cells
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Advantages of finite cell lines
\-creates large population of similar cells
\-most cellular characteristics are maintained
\-can transform cells to grow indefinitely
\-most cellular characteristics are maintained
\-can transform cells to grow indefinitely
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Disadvantages of finite cell lines
\-cells have a tendency to differentiate over time in culture
\-culture tends to select to aberrant (cancerous) cells
\-culture tends to select to aberrant (cancerous) cells
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Continuous cell lines
\-can be subcultures indefinitely
\-can happen naturally or artificially
\-can happen naturally or artificially
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HeLa cells
\-cervical cancer cells removed from Henrietta Lacks
\-grown in culture and used extensively in science
\-grown in culture and used extensively in science
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Continuous cell lines advantages
\-easy to maintain in culture
\-easy to obtain large population of cells
\-typically easy to manipulate gene
\-easy to obtain large population of cells
\-typically easy to manipulate gene
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Continuous cell lines disadvantages
\-changes over time
\-function may be different than in Vito cells
\-function may be different than in Vito cells
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Transformed, infinite or established cells
\-changed from normal cells to cells with properties of cancer
\-mutates indiscriminately
\-cell has altered functional, morphological, and growth characteristics
\-mutates indiscriminately
\-cell has altered functional, morphological, and growth characteristics
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Transformation
\-spontaneous or induced mutation resulting in a different phenotype
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Transfection
introduction of DNA into a cell
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Suspension cells (anchorage-independent)
\-Growing in suspension
\-cell lines derived from blood
\-cell lines derived from blood
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Adherent or monolayer cells
\-must bind to solid surface to survive and propagate
\-growing as a monolayer attached to tissue culture flask
\-cells derived from solid tissue, endothelial, epithelial, neuronal, fibroblasts
\-growing as a monolayer attached to tissue culture flask
\-cells derived from solid tissue, endothelial, epithelial, neuronal, fibroblasts
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Advantages of adherent growth
\-cells adhere well
\-allows studies to be more easily performed
\-allows studies to be more easily performed
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Suspension growth advantages
\-large numbers of cells
\-easy to harvest
\-easy to harvest
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Steps to grow cells in culture
1. Place cells in culture
2. Grow cells successfully
3. Add trypsin to separate cells
4. Repeat
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Confluence
No more space for cells to grow
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What does cell growth and differentiation depend on?
\-nature of cells
\-substrate
\-culture medium
\-incubation temp
\-cell-cell and cell-matrix interaction
\-substrate
\-culture medium
\-incubation temp
\-cell-cell and cell-matrix interaction
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Promotion of cell proliferation
\-low cell density
\-growth factors
\-growth factors
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Inhibition of cell proliferation
\-density limitation (high cell density)
\-contact inhibition
\-signals from environment (tumor suppressor genes)
\-contact inhibition
\-signals from environment (tumor suppressor genes)
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Contact inhibition
Cells stop dividing when they come in contact with each other
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Cell adhesion
\-Important for cell proliferation and differentiation
\-involves cell-cell and cell-matrix interactions
\-involves cell-cell and cell-matrix interactions
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Cell-cell interactions
\-CAMs (cell adhesion molecules) and cadherins
\-have a tight junctional complex
\-have a tight junctional complex
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Cell-matrix interactions
\-Integrin, transmembrane proteoglycan
\-promotes cell proliferation and differentiation
\-promotes cell proliferation and differentiation
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Factors affecting cell culture
\-appropriate cells
\-suitable environment
\-suitable environment
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Solid phase
\-nontoxic biologically inert substance
\-surface treated by coated with matrix substrate and feeder layers
\-surface treated by coated with matrix substrate and feeder layers
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Feeder layer
Giving nutrients and solid layer to grow on
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Liquid phase
\-components of culture media
\-inorganic salts
\-carbohydrates
\-protein and peptides
\-amino acids
\-fatty acids and lipids
\-vitamins
\-trace elements
\-inorganic salts
\-carbohydrates
\-protein and peptides
\-amino acids
\-fatty acids and lipids
\-vitamins
\-trace elements
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Inorganic salts
\-retain osmotic balance of cells
\-regulate membrane potential
\-required in cell matrix for cell attachment and enzyme cofactors
\-regulate membrane potential
\-required in cell matrix for cell attachment and enzyme cofactors
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Carbohydrates
Main source of energy from glycolysis
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Proteins and peptides
\-used to replace those normally present in serum
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Amino acids
Important for cell proliferation and differentiation
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Fatty acids and lipids
Important in serum free media
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Vitamins
\-precursors for numerous co-factors
\-thiamine, riboflavin, and biotin commonly used
\-thiamine, riboflavin, and biotin commonly used
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Vitamin B
Necessary for cell growth and proliferation
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Trace elements
\-zinc, copper, selenium, tricarboxylic acid
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Buffering systems
\-optimal pH is 7.2-7.4
\-yellow = acidic
\-purple = basic
\-red = neutral
\-yellow = acidic
\-purple = basic
\-red = neutral
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Serum
\-mix of albumins, growth factors and inhibitors
\-increase buffering capacity
\-able to bind and neutralize toxins
\-can help reduce risk of contamination
\-increase buffering capacity
\-able to bind and neutralize toxins
\-can help reduce risk of contamination
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Gaseous phase
\-carbon dioxide
\-oxygen
\-oxygen
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Carbon dioxide
important for buffering systems
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Oxygen
\-most cells in culture require low o2
\-anaerobic glycolysis
\-high o2 can be toxic
\-anaerobic glycolysis
\-high o2 can be toxic
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Optimum temperature
\-body temp of animals
\-anatomical variation of temperature (skin differs)
\-anatomical variation of temperature (skin differs)
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Aseptic techniques
\-antibiotics
\-improvement of laboratory condition
\-aseptic techniques
\-improvement of laboratory condition
\-aseptic techniques
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Hayflick’s phenomenon
\-cells will stop dividing and eventually die even with proper nutrients
\-correlation between maximum number of passages and aging
\-correlation between maximum number of passages and aging
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Progeria
defects that cause premature aging
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Cancer cells
\-immortal
\-will continue to grow and create multiple layers of cells
\-will continue to grow and create multiple layers of cells
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Growth cycle
1. Lag phase
2. Log phase
3. Plateau phase
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Lag phase
\-cells adjusting to new environment
\-no growth
\-no growth
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Log phase
\-exponential increase in cells
\-transfer to subculture
\-optimal time for sampling
\-transfer to subculture
\-optimal time for sampling
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Plateau phase
\-becomes confluent (no room)
\-growth rate reduced
\-growth rate reduced
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Cryopreservation
Prevents growth and mutation
Note 
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