Introduction to animal cell culture
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Transcript of Introduction to animal cell culture
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Introduction to Animal Tissue culture
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What is tissue culture? In vitro culture (maintain and/or proliferate) of cells,
tissues or organs.
Types of tissue culture
• Cell culture
• Primary explant culture
• Organ culture
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Cell culture
Explant
culture
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Cell culture:Adherent monolayer on a solid substrate (various cell types) suspension in the culture medium (few cell types)
Primary explant culture:A fragment of tissue attachment and migration occurs in the plane of the solid substrate
Organ culture:A spherical or three-dimensional shape specific histological interaction
Three major categories of tissue culture
Explant: living cells, tissues, or organs from animals or plants that transfer to a nutrient medium.
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Cell culture & Enzymatic Dissociation
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Tissue from an explant is dispersed, mostly enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture.
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Advantages & Disadvantages
Advantages Development of a cell line over several generations Scale-up is possible Absolute control of physical environment Homogeneity of sample Less compound needed than in animal models
Disadvantages Cells may lose some differentiated characteristics. Hard to maintain Only grow small amount of tissue at high cost Dedifferentiation Instability, aneuploidy
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Tissue Culture
Is the growth of tissues or cells separate from the organism.
This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar.
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Advantages Some normal functions may be maintained. Better than organ culture for scale-up but not ideal.
Disadvantages Original organization of tissue is lost.
Advantages & Disadvantages
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Organ culture The entire embryos or organs are excised from the body and
culture
Advantages Normal physiological functions are maintained. Cells remain fully differentiated.
Disadvantages Scale-up is not recommended. Growth is slow. Fresh explantation is required for every experiment.
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EMP04 9
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Why do we need Cell culture?
Research◦ To overcome problems in studying cellular behavior such as:
confounding effects of the surrounding tissues variations that might arise in animals under experimental
stress◦Reduce animal use
Commercial or large-scale production◦ Production of cell material: vaccine, antibody, hormone
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Initiation of culture
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Tissue
Primary culture
Cell line Continuous cell line
Subculture
Stored Stored
Animal Plant
Finite numbers Indefinite numbers
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Types of Cell culture
1. Primary Cultures
Derived directly from excised tissue and cultured either as:
Outgrowth of excised tissue in culture
Dissociation into single cells (by enzymatic digestion or mechanical dispersion).
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Primary Culture
Preparation
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Characteristics of Primary Cultures
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Primary Culture
Preparation
Characteristics:
Morphologically similar to the parent tissue
Limited number of cell divisions
Best experimental models for in vivo situations
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Advantages & Disadvantages◦ Advantages:
usually retain many of the differentiated characteristics of the cell in vivo
◦ Disadvantages:
initially heterogeneous but later become dominated by fibroblasts.
the preparation of primary cultures is labor intensive
can be maintained in vitro only for a limited period of time.
Difficult to obtain
Relatively short life span in culture
Very susceptible to contamination
May not fully act like tissue due to complexity of media14
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Types of Cell culture2. Continuous Cultures
derived from subculture (or passage, or transfer) of primary culture Subculture = the process of dispersion and re-culture the cells after they have increased to occupy all of the available substrate
in the culture
usually comprised of a single cell type can be serially propagated in culture for several passages
There are two types of continuous cultures Cell lines Continuous cell lines
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Types of continuous culture1) Cell lines
Cell lines derived from primary cultures have a limited life span
After the first subculture, the primary culture becomes cell line
finite life, senesce after approximately thirty cycles of division
usually diploid and maintain some degree of differentiation
it is essential to establish a system of Master and Working banks in order to maintain such lines for long periods
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Types of continuous culture2) Continuous cell lines
can be propagated indefinitely
generally have this ability because they have been transformed by:
tumor cells.
viral oncogenes
chemical treatments
Spontaneously
the disadvantage of having retained very little of the original in
vivo characteristics 17
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Transformation VS Transfection
Transformation◦Spontaneous or induced permanent phenotypic changes
resulting from change in DNA and gene expression that result and effect in: growth rate mode of growth (loss of contact inhibition) specialized product formation longevity loss of need for adhesion
Transfection◦ Introduction of DNA into a cell (like viral DNA)
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Cell Culture Morphology
Morphologically cell cultures take one of two forms:◦ growing in suspension (as single cells or small free-floating clumps)
cell lines derived from blood (leukemia, lymphoma)◦ growing as a monolayer that is attached to the tissue culture flask.
Cells from solid tissue (lungs, kidney, breast), endothelial, epithelial, neuronal, fibroblasts
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Hela-Epithelial
MRC5-Fibroblast SHSY5Y-Neuronal
BAE1-Endothelial MCF-7 breast
HT1080- kidney 3LL - lungs
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• Excellent model systems for studying:The normal physiology and biochemistry of cells
The effects of drugs and toxic compounds on the cells
Mutagenesis and carcinogenesis
• Used in drug screening and development
• Large scale manufacturing of biological compounds
(vaccines, insulin, interferon, other therapeutic protein)
Cell culture application