Animal Cell Biology Study Notes PDF

What defines a cell line? - Cells with proliferate potential and sometimes indefinite - They arise from primary cell cultures What defines a cell strain? - sub population and selection of cell line through genetic manipulation What is known as a primar...

What defines a cell line? - Cells with proliferate potential and sometimes indefinite - They arise from primary cell cultures What defines a cell strain? - sub population and selection of cell line through genetic manipulation What is known as a primary cell culture? - Morphologically similar to the parent tissue - Capable of only a limited number of cell division; they are also susceptible to contact inhibition, that is, they will stop growing when they have reached confluency What is known as a cell culture? - Cells are removed from the tissue directly and disaggregated by enzymatic or mechanical means What is a histotypic culture? - Cells that have been re-aggregated to create a 3D structure; aims to attain the density of cells found in the original tissues - Ways to recombine: perfused multilayer from monolayer; spheroids or organoid; sponge or scaffold; filter well insert; perfused capillary bed What is organotypic culture? - Same procedures with histotypic culture but recombining cells with different lineages, e.g. epidermal keratinocytes and dermal fibroblasts How many types of cell lines are there? What are their differences? Finite Cell Line Continuous cell line Usually divide only a limited number of times Has unlimited proliferative potential May acquire a stable, heritable mutation, giving Undergone genetic alterations & their behaviour rise to continuous cell line in vitro may not represent the in vivo situations - Frequently correlates with tumorigenicity What types of cells are there? Adherent Cells Suspension Cells Cells that grow while adhering the surface of the culture - In a suspended state vessels - Can survive & proliferate w/o being attached to a substratum - Fibroblasts - Hematopoietic cells - Epithelial cells - Transformed cell lines - Mesenchymal stem cells (MSCs) How should we choose the choice of disaggregation? 1. When small amounts of tissue are available a. Dissection of primary explant 2. More tissue are available but high recovery is required a. Enzymatic disaggregation 3. Large amount of soft tissue are available and size of yield is not important a. Mechanical disaggregation What are the advantages and disadvantages of the fine dissection method? Advantages: Useful for small amount of tissue; avoid risk of losing cells during mechanical/enzymatic disaggregation Disadvantages: Poor adhesiveness of some tissue; Require the selection of more migratory cells in outgrowth What are the advantages and limitations in enzymatic disaggregation? Advantages: avoid problems of selection by migration; yield higher number of cells that are more representative of a while tissue in a shorter time Disadvantages: Trypsin may damage cells upon prolonged exposure; select for protease stress-resistance cells What are the major advantages of cell culture? The ability to control: - Physiochemical: temp, pH, osmotic pressure, O2 and CO2 tension - Physiological environment: Hormone & nutrients What is known as a complete media? - Contain large number of diff amino acid, vitamins, extra metabolites (nucleoside, TCA, cycle intermediates, lipids) & minerals 4.5 g/L glucose - high level of glucose Phenol red - pH indicator NaHCO3 - sodium bicarbonate - pH regulator What are the basic components of a media? 1. Inorganic salts 2. Amino acids 3. Carbohydrates 4. Protein & peptides 5. Fatty acids & lipids 6. Vitamin & trace elements 7. Serum 8. Antibiotics What is the purpose of adding phenol red? What is the disadvantages of it? - pH indicator which allows constant monitoring of pH - Low pH, turn medium yellow; high pH, turn medium purple Disadvantages: similar structure to oestrogen; may trigger memory of breast cancer cell line; unnecessary activation What is the purpose of adding amino acid? What are the disadvantages of it? - L-glutamine, as an essential amino acid, provides nitrogen for NAD, NADPH & nucleotides, & serves as secondary energy source for metabolism Disadvantages: in excess, will produce ammonia, which is toxic to the cells What are the advantages and disadvantages of adding serum? - A rich source of a protein & includes albumin, transferrin, aprotinin, fetuin and fibronectin Advantages Disadvantages Provides several growth factors & hormones Batch variation including in growth promotion & specialized cell function Contains several binding proteins which can carry Testing needed to ensure quality of each batch other molecules into the cells Promote attachment of cells to the substrate. It Increase the risk of contamination also provides spreading factors that help the cells to spread out before they begin to divide Provide protease inhibitors which protects cells Complicates downstream process; purification & from proteolysis isolation of cell culture products Increases the viscosity of medium, thus protect May contains some growth inhibiting factors cells from mechanical damages during agitation of suspension cultures What is the purpose of cryopreservation? - To enable stocks of cells to be stored to prevent the need to have all cell lines in cultures at all times What is the basic principle of cryopreservation? - The rate of cooling is optimized to allow time for intracellular water to have all cell lines in culture at all times - Addition of cryoprotectant to the cells depresses the temperature at which intracellular ice is formed & allows cooling rates to be reduced for more efficient water loss. When should we do cryopreservation and how should we do it? - Log phase; 80% before stationary; maximum viability - Resuspend with cryoprotectant (DMSO or glycerol); slow cooling