Background

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Chapter 1

• Introduction

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Background

• Tissue culture - 20th century
• Method to study behavior of animal cells
• No systemic variations that arise in vivo (in the
  organism)
• From homeostasis
• Experimental stress
• Source
• Undisaggregated fragments of tissue
• Hence the name
• Todays sources
• Dispersed cell cultures

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Background

• Isolation of cells-old way
• Mostly by surgical subdivision of culture
• First was L929 from mouse L-Cells

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Background

• Isolation-standard way
• Trypsin
• Used to re-suspend cells adherent to the cell
  culture dish wall during the process of
  harvesting cells
• Other aids to tissue/cell culture
• Antibiotics
• Facilitates long-term cell line propagation
• May harbor antibiotic-resistant contamination
• Defined media
• Led to development of serum-free media
• Eliminated unwanted contaminations
• Eliminates safety problems
• Eliminates unwanted proteins

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Background

• Tissue culture terms-Organ culture vs. Cell
  culture
• Organ culture
• 3D culture of undisaggregated tissue
• Retains some/all histological features of the
  tissue in vivo
• Cell culture
• Derived from dispersed cells taken from
• Original tissue
• Primary culture
• Cell line
• Cell strain
• Enzymatic, mechanical, or chemical disaggregation
• Other terms
• Histotypic culture
• Cells reaggregated/grown to recreate a 3D
  structure w/tissue-like density
• Organotypic
• Recombination of cells of different lineages
• Epidermal keratinocytes with dermal fibroblasts

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Background

• Early tissue sources
• Frog (1907)
• Cold blooded so incubation not required
• Tissue regeneration more common in lower
  vertebrates
• Not similar to higher vertebrates
• Warm-blooded dont regenerate
• Needed model closer to humans
• Rodents
• Produces continuous cell lines
• Can establish transgenic cells

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Background

• Human cells
• Optimum for research
• Difficult to culture
• Ethics
• Use tumor cells
• HeLa cells
• Lower vertebrates and invertebrates
• Largely ignored except for unique features
• Regeneration in amphibians
• Metamorphosis in insects
• Pest control
• Toxicity and virology studies in insects
• May be useful as vectors

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This slide demonstrates the use of mosquito cell
cultures to detect dengue virus. The results for
this patient's blood sample are positive the
fluorescing cells seen here are infected with
dengue virus. Source CDC
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Background

• Investigations using tissue culture
• Intracellular activity
• DNA transcription, protein synthesis, energy
  metabolism, drug metabolism, cell cycle,
  differentiation, apoptosis
• Intracellular Flux
• RNA processing, hormone receptors, metabolite
  flux, calcium mobilization, signal transduction,
  membrane trafficking

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Background

• Investigations using tissue culture
• Cell products
• Proteomics, secretion, biotechnology, bioreactor
  design, product harvesting, down-stream
  processing
• Immunology
• Cell surface epitopes, hybridomas, cytokines and
  signaling, inflammation

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Background

• Investigations using tissue culture
• Genomics
• Genetic analysis, transfection, infection,
  transformation, immortalization, senescence
• Tissue Engineering
• Tissue constructs, matrices and scaffolds, stem
  cell sources, propagation, differentiation
• Toxicology
• Infection, cytotoxicity, mutagenesis,
  carcinogenesis, irritation, inflammation

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Background

• Investigations using tissue culture
• Cell-Cell interaction
• Morphogenesis, paracrine control, cell
  proliferation kinetics, metabolic cooperation,
  cell adhesion and motility, matrix interaction,
  invasion
• Pharmacology
• Drug action, ligand receptor interactions, drug
  metabolism, drug resistance

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Advantages of Tissue Culture

• Control of the Environment-two major advantages
• Control of physiochemical environment
• pH, temp, osmotic pressure, and O2 and CO2
  tension (gas exchange)
• Very precise control
• Control of physiological conditions
• Can be controlled but NOT defined
• Aids in addition of supplements to media
• Caused by variations in batches
• Contain undefined elements e.g. hormones
• Aids in understanding undefined needs of the
  cell/tissue

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Advantages of Tissue Culture

• Characterization and Homogeneity of Sample
• Samples invariably are heterogeneous
• Replicates difficult
• Very in constituent types
• After one or two passages
• Usually become uniform from
• Random mixing of the cultures
• Selective pressures of the culture conditions
• . after each subculturing (passage)
• Replicate become identical
• Perpetuates characteristics of the cell line for
  several generations/indefinitely (if cell line
  stored in liq. N2)
• -Reduces need for statistical analysis of
  variance
• -preserved stocks can be validated for future
  research

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Advantages of Tissue Culture

• Economy, Scale, and Mechanization
• Cultures exposed to reagents directly
• Lowers reagent requirement/costs
• Not lost by excretion
• Not lost to distribution to tissues NOT under
  study
• Avoids ethical questions
• Animal experimentation

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Limitations

• Expertise
• Techniques carried out under strict aseptic
  conditions
• Contaminants like bacteria, molds, and yeasts
  occur
• Animal cells grow slowly
• Complex environments for growth
• Cells not found in isolated environments
• .do not sustain independent existence without
  the complex environment simulating blood
  plasma/interstitial fluid
• Increases need for operator skill to recognize
  problems
• Avoidance of cross-contamination

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Limitations

• Quantity
• Expense of effort and materials for small amount
  of tissue
• Levels of production
• 1-10 g of cells
• 2 or three people ding the culturing
• 10-100 g
• Little more effort of a larger lab
• 100 g and above
• Pilot-plant scale
• Cost of producing cells
• About 10 x the cost of using animal tissue
• gt 10 g tissue EXPENSIVE
• Reasons must be COMPELLING!

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Limitations

• Dedifferentiation and Selection
• Early work showed increase of loss of phenotypic
  characteristics
• Thought to be from lack of differentiation
• Shown to be largely due to overgrowth of
  undifferentiated cells
• Led to development of serum-free selective media
• Allows for isolation of specific lineages
• Can restore differentiated properties

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Limitations

• Origin of Cells
• If you lose differentiated properties
• Difficult to relate cultured cells to functional
  cells
• Instability
• Major problem with continuous cell lines
• Heterogeneity in growth rate/capacity to
  differentiate
• Can produce variability from one passage to the
  next

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Types of tissue Culture

• Three methods
• Organ culture (25.2)
• The characteristic of the tissue in vivo is
  retained (in part) in the culture
• Do not grow rapidly
• Requires fresh explantations
• Greater effort
• Poorer reproducibility of the sample than with
  cell culture
• Quantitation
• More difficult
• Amount of material limited, . effort for
  dissection GREATLY increased
• DO retain specific histological interactions
• Tissue cultured at a liquid-gas interface
• On a raft, grid, or gel
• Supports a spherical/3D shape
• Histological structure maintained

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Types of tissue Culture

• Three methods
• Primary explant culture (12.3.1)
• Fragment of tissue placed at a plastic-liquid
  interface
• Migration promoted in the plane of the solid
  substance (after attachment)
• Formation of cell lines implies
• Increase in total number of cells over several
  generations
• Ultimate predominance of cells/cell lineages with
  high growth capacityresults in
• High degree of uniformity in the cell population

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Intestinal Explant Culture
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Types of tissue Culture

• Three methods
• Cell culture
• Tissue/outgrowth from primary explant (12.3,
  13.7)
• Dispersed into cell suspension
• Mechanically
• Enzymatically
• May be derived from primary explants or dispersed
  cell suspensions
• Enhances propagation of cell lines

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Types of tissue Culture

• Other terms
• Monolayer
• Implies that the cells will attach
• Common to most normal cells
• Suspension cultures
• Derived fro cells that can survive/proliferate
  w/o attachment
• Restricted to
• Hematopoietic cells
• Transformed cell lines
• Cells from malignant tumors

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Types of tissue Culture

• Subculture of cells or cell lines
• Advantages
• Propagation
• More cells
• Possibility of cloning
• Increased homogeneity
• Characterization of replicate samples
• Frozen storage

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Types of tissue Culture

• Subculture of cells or cell lines
• Disadvantages
• Trauma of enzymatic or mechanical disaggregation
• Selection of cells adapted to culture
• Overgrowth of unspecialized or stromal cells
• Genetic instability
• Los of differentiated properties
• Increased risk of misidentification or
  cross-contamination

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