Sart Exam 2 - Cell Culture

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Title: Three Ages of Woman
Artist: Gustav Klimt

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Tissue Culture

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-removal of cells, tissues, or organs from an animal or plant and their subsequent placement into an artificial environment conducive to growth
-used to prepare finite or continuous cell cultures
-similar biochemically & physiologically to parent tissue

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Organ Culture

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-culture of whole organs or intact organ fragments w/intent of studying their continued function or development
-maintained for hrs-days by perfusion w/oxygenated blood or serum
-used for metabolism & drug studies
-provides most accurate reflection of organism's physiology

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Cell Culture

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-cells are removed from organ fragments prior to or during cultivation; disrupts their normal relationships w/neighboring cells
-explains non- "in vivo" experiments
-non-tissue growth of plant and animal cells

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Mammalian Cell Culture

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-Eukaryotic cells are must more difficult to culture than most prokaryotes
-demand complex media
-very susceptible to contamination & overgrowth by microbes (bacteria, yeasts, fungi)

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Why we need Cell Culture?

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1. RESEARCH
-overcome problems in studying cellular behavior
confounding effects of surrounding tissues;
variations that might arise in animals under exp.
stress);
-reduces animal use
2. COMMERICIAL or LARGE SCALE PRODUCTION
-production of cell material: vaccine, antibody,
hormone

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Two Types of Cell Culture

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1. DERIVED DIRECTLY from excised tissue & cultured either as outgrowth of excised tissue in culture, or dissociation into single cells (by enzymatic digestion or mechanical dispersion)
2. DERIVED FROM SUBCULTURE / CONTINUOUS / INFINITE (passage or transfer) of primary culture; usually comprised of single cell type; can be serially propagated in culture for several passages

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Subculture

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-the process of dispersion and re-culture the cells after they have increased to occupy all of the available substrate in the culture

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Primary Cell Culture

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1. from outgrowth of migrating cells from piece of tissue or tissue that's disaggregated by enzymatic, chemical, or mechanical methods
2. formed from cells that survive disaggregation process, attach to cell culture vessel (or survive in suspension) and proliferate
3. morphologically similar to parent tissue - same karyotype
4. capable of limited # of cell divisions - enter senescence - die out
5. more physiologically similar to in vivo cells
6. generally more diff. than culture of continuous cell lines
7. not 'de-differentiated'

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Senescence

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-non-proliferative state
-stop dividing
-eventually die out

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Advantages of Primary Cell Culture

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-represent best experimental models for in vivo situations
-no de-differentiated
-same karyotype

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Disadvantages of Primary Cell Culture

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-difficult to obtain
-relatively short life span in culture
-very susceptible to contamination
-may not fully act like tissue due to complexity of media
-considerable variation in population & between preparations

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Tumor Primary Cell Culture

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-easier to create as tumor cells cycle/growth regulations have been altered
-tumor cells often produce own growth factors (autocrine)

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Finite Cell Cultures

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-technically continuous
-secondary or sub-clone culture
-formed after first subculturing (passaging) of primary cell culture
-will proliferate for limited # of cell divisions - senescence
-factors which control replication of such cells in vitro are related to degree of differentiation of cell
-easier than use of primary cell cultures, especially for generation of stably transfected clones

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Advantages of Finite Cell Culture

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-can obtain large population of similar cells
-most cellular characteristics are maintained
-can transform cells to grow indefinitely

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Disadvantages of Finite Cell Culture

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-cells have tendency to differentiate over time in culture
-over time the culture tends to select for ABERRANT CELL (not very close to parent cell)

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Continuous Cell Lines

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-cell has demonstrated potential to be sub-cultured indefinitely
-infinite cell line/immortal cells - transformed cells
-generally easier to work w/than primary or finite cell cultures
-cells have undergone genetic alterations - may not represent in vivo situation

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Infinite Cell Line - Immortal Cells/Transformed Cells

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-cells who's growth properties have been altered
-finite cell cultures will eventually either die out or acquire stable, heritable mutation that gives rise to continuous cell line capable of unlimited proliferative potential
-alteration commonly known as in vitro transformation or immortalization & frequently correlated w/tumorigenicity

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HeLA Cells

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-HeLa cells = Henrietta Lacks
-human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18)

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Advantages of Continuous Cell Lines

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-easy to maintain in culture
-easy to obtain large population of cells
-typically easy to manipulate gene expression

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Disadvantages on Continuous Cell Lines

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-the more aggressive the cell line = the more it changes over time in culture
-not clear how the function of these cells relates to that of other cells, healthy or diseased

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Transformation

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=changed from normal cells to cells w/many of the properties of cancer cells
=genetic alteration can grow forever
-chemical or gamma ray treated cells can become infinite w/loss of growth factors
-viral infection w/SV40 T antigen can insert oncogenes & lead to p58 and RB gene alteration
-no matter how transformation occurred, the result is a cell w/altered functional, morphological, & growth characteristics

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Transfection

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-'transformation' in bacteria
-introduction of DNA into a cell (like viral DNA)
-initiation of culture

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Cell Culture Morphology

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-morphologically cell cultures take two forms:
1. Suspension Cells: (anchorage-independent)
-growing in suspension as single cells or small
clumps
-cell lines derived from blood (leukemia,
lymphoma)
2. Adherent or monolayer cells:
-must bind to solid surface to survive & propagate
-growing as a monolayer that is attached to tissue
culture flask
-cells DERIVED FROM SOLID TISSUE (lungs, kidney),
endothelial, epithelial, neuronal, fibroblasts

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Adherent Cells

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-highly anchorage-independent & adhere lightly even to tissue culture dishes
-to survive & grow most cells require a surface they can attach to

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Suspension Cell Cultures

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-do not require attachment for cell proliferation
-growth of cells in tissue culture dishes looks more haphazard than the growth of anchorage dependent cells w/cells only loosely attached to the surface
-some cells can survive & divide while being suspended in a fluid media & stirred/shaken

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Advantages of Adherent Growth

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-cells very easy to deal/work with
-ability of the cells to adhere & spread on surfaces such as coverslips, making microscopy, hybridizations, & functional assays more easily performed

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Advantages of Suspension Growth

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-large numbers of cells that can be achieved
-ease of harvesting - can grow in large batches

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Growing Cells in Culture

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1. place cells in culture dish
2. give nutrients, growth factors, keep free from bacteria
3. cells will grow to cover surface of dish
4. can take cells out of this culture & start new culture
5. SPLITTING CELLS from one dish to another is a passage

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Biology of Culture Cells: Cell Growth & Differentiation in Culture Depends on:

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1. nature of cells
2. culture environment
3. nature of the substrate on which cells grow
4. physicochemical & physiological constitution of culture medium
5. constitution of gas phase
6. incubation temperature
7. cell-cell and cell-matrix interaction

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Biology of Culture Cells: Factors Affecting Cell Proliferation:

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1. promotion of cell proliferation - low cell density (leaves the cell w/free edge)
2. inhibition of cell proliferation - density limitation: high cell density - contact inhibition

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Biology of Culture Cells: Factors Affecting Cell Adhesion:

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-matrix coated surface promotes cell proliferation & differentiation
1. cell-matrix interaction: integrin, transmembrane proteoglycan
2.cell-cell interaction: tight junctional complex in epithelial cells

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Biology of Culture Cells: Factors Affect Cell Culture Success:

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1. Solid Phase: substrate or phase on which cells grow
2. Liquid Phase: Components of culture media
3. Gaseous Phase
4. Temperature: depends on body temp of animals from which they were obtained
5. Aseptic techniques: prevention of contamination

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Factors Affect Cell Culture Success: SOLID PHASE

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-substrate or phase on which the cells grow (glass, plastic, collagen, agar)
-anchorage dependent cells require a nontoxic, biologically inert to attach and allow movement for growth
-most convenient vessels are polystyrene plastic
-coated w/matrix substrate (collagen, poly-l-
lysine, matrigel)
-feeder layers: monolayer of supporting cells,
perhaps promote cell growth & differentiation
by cell contact & substrate secreted

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Factors Affect Cell Culture Success: LIQUID PHASE

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1. Inorganic Salts: retain osmotic balance of cells &
signal enzymes
2. Carbohydrates: main energy source from glycolysis
3. Proteins & Peptides: replace those normal present
in serum; building
4. Amino Acids: imp. for cell proliferation &
differentiation
5. Fatty Acids & Lipids: cholesterol & steroids
essential for specialized cells
6. Vitamins: vitamins B are necessary for cell growth
& proliferation
7. Trace Elements: zinc, copper, selenium &
tricarboxylic acid intermediates
8. Buffering Systems: most cells need optimal pH
conditions in 7.2-7.4 range
9. Osmolarity: similar to plasma osmolarity 290
mOsm
10. Serum: undefined factors: complex mix of albumins, growth factors & growth inhibitors; bacterial like serum as well, so it must be sterile

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Selenium

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-is a detoxifier
-helps remove oxygen free radicals - can cause chaos!

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Factors Affect Cell Culture Success: GASEOUS PHASE

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-carbon dioxide: important for buffering system - 5 to 10% CO2
-oxygen - most cells in culture require low oxygen tension

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Number of Cell Divisions

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-normal cells have a limit to # of times can be passed in culture: vary from cell-cell type; common limit 40-60 passages

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Hayflick's Phenomenon and Aging

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-# of passages decreases (when age increases) when cells are harvested from older individuals
-Progeria

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Progeria

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-collection of defects which causes premature aging
-genetic disorder - physical symptoms like gray hair, wrinkled skin, hair loss, muscle degeneration
-cells from these individuals show dramatically decreased passage #

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Contact Inhibition

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-normal animal cells STOP dividing when they come into contact w/one another
-these cells can be triggered to begin dividing again by giving again by giving them more room
-cancer cells appear to be immortal, do not display contact inhibition

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Growth Cycle in Attachment Culture

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-growth cycle is typically divided into three phases
1. Lag Phase
2. Log Phase
3. Plateau Phase

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Lag Phase

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-time following subculture & reseeding during which there is little evidence of an increase in cell number
-PERIOD OF ADAPTION

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Log Phase

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-take cells for experiment from this phase
-period of EXPONENTIAL GROWTH - increase in cell # following lag period & terminating one or two doublings after confluence is reached
-depends on seeding density, growth rate of cells, & density at which cell proliferation is inhibited by density
-optimal time for sampling b/c population at most uniform & viability is high

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Plateau Phase

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-toward end of log phase culture becomes confluent
-growth rate of culture is reduced, & in some cases, cell proliferation eases almost completely after one or two further population doublings - growth fraction falls to between 0-10%

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Cryopreservation of Cell Lines

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-store on ice/dry ice - freeze cells
-aim is to enable stocks of cells to be stored to prevent need to have all cells lines in culture at all times
-reduced risk of microbial contamination
-reduced risk of cross contamination w/other cell lines
-reduced risk or genetic drift & morphological changes
-work conducted using cells at consistent passage #
-reduced costs (consumables & staff time)

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The Dilemma - passage # or how long can cells be used?

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-high number of subculturing will change cell biochemical & molecular properties, morphology, response to agonists, growth rates & other responses
-may be cell response to changed conditions from tissue
-when doubling times significantly change, maybe now they are a 'different strain' then stated with