Sinh học - Introduction to animal tissue culture

Introduction to Animal Tissue cultureWhat is tissue culture? In vitro culture (maintain and/or proliferate) of cells, tissues or organs. Types of tissue cultureCell culturePrimary explant cultureOrgan culture2Cell cultureExplant cultureOrgan culture3Cell 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 substrateOrgan culture:A spherical or three-dimensional shape specific histological interactionThree major categories of tissue culture Explant: living cells, tissues, or organs from animals or plants that transfer to a nutrient medium.Cell culture & Enzymatic Dissociation4Tissue from an explant is dispersed, mostly enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture.Advantages & DisadvantagesAdvantages 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, aneuploidy5Tissue 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.67AdvantagesSome normal functions may be maintained. Better than organ culture for scale-up but not ideal.DisadvantagesOriginal organization of tissue is lost. Advantages & DisadvantagesOrgan cultureThe entire embryos or organs are excised from the body and cultureAdvantagesNormal physiological functions are maintained. Cells remain fully differentiated. DisadvantagesScale-up is not recommended. Growth is slow. Fresh explantation is required for every experiment.8EMP049Why do we need Cell culture?ResearchTo overcome problems in studying cellular behavior such as:confounding effects of the surrounding tissues variations that might arise in animals under experimental stressReduce animal useCommercial or large-scale productionProduction of cell material: vaccine, antibody, hormone10Initiation of culture11TissuePrimary cultureCell lineContinuous cell lineSubcultureStoredStoredAnimalPlantFinite numbersIndefinite numbersTypes of Cell culturePrimary CulturesDerived directly from excised tissue and cultured either as:Outgrowth of excised tissue in cultureDissociation into single cells (by enzymatic digestion or mechanical dispersion).12Primary Culture PreparationCharacteristics of Primary Cultures13Primary Culture Preparation Characteristics:Morphologically similar to the parent tissueLimited number of cell divisionsBest experimental models for in vivo situationsAdvantages & DisadvantagesAdvantages:usually retain many of the differentiated characteristics of the cell in vivoDisadvantages:initially heterogeneous but later become dominated by fibroblasts. the preparation of primary cultures is labor intensivecan be maintained in vitro only for a limited period of time.Difficult to obtainRelatively short life span in cultureVery susceptible to contaminationMay not fully act like tissue due to complexity of media14Types of Cell cultureContinuous Culturesderived 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 typecan be serially propagated in culture for several passagesThere are two types of continuous culturesCell linesContinuous cell lines15Types of continuous cultureCell linesCell lines derived from primary cultures have a limited life spanAfter the first subculture, the primary culture becomes cell line finite life, senesce after approximately thirty cycles of divisionusually diploid and maintain some degree of differentiationit is essential to establish a system of Master and Working banks in order to maintain such lines for long periods 16Types of continuous culture2) Continuous cell linescan be propagated indefinitely generally have this ability because they have been transformed by:tumor cells. viral oncogenes chemical treatmentsSpontaneously the disadvantage of having retained very little of the original in vivo characteristics 17Transformation VS TransfectionTransformationSpontaneous 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 longevityloss of need for adhesion TransfectionIntroduction of DNA into a cell (like viral DNA)18Cell Culture MorphologyMorphologically 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 19Hela-EpithelialMRC5-FibroblastSHSY5Y-NeuronalBAE1-EndothelialMCF-7 breastHT1080- kidney3LL - lungs Excellent model systems for studying:The normal physiology and biochemistry of cellsThe effects of drugs and toxic compounds on the cellsMutagenesis and carcinogenesis Used in drug screening and development Large scale manufacturing of biological compounds (vaccines, insulin, interferon, other therapeutic protein)Cell culture application