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ANIMAL CELL AND TISSUE CULTURE Principles Primary cells, cell lines • and immortalized cells Media for cell cultivation Cell growth and metabolism Cell characterization and preservation Animal Cell Growth Growth: Increase of cell numbers during time in culture Characteristic Growth Curve (mostly anchorage-dependent cells) Lag Phase upon seeding Exponential Growth Phase Plateau Phase (saturation cell density) Cells become confluent, occupying all available growth surface; Contact inhibition: inhibition of cell motility, membrane ruffling… Limited proliferation can also be due to nutrient, growth factor depletion and metabolite accumulation (limiting factor for suspension cells) Adapted from: Palsson, B.Ø. and Bhatia, S.N., Tissue Engineering, Pearson Prentice Hall Bioengineering, 2004 Cell Growth Terminology Growth parameters: Viable cell concentration, nv Fraction of viable cells, fv Specific growth rate, µ dn = increase in cell number dt = time interval n = cell number Doubling time, Dt (i.e. time for a population to double in number) Population doubling time, PD (i.e. total number of population doublings of a cell line since its initiation in vitro) Specific death rate, kd 1 dn µ= n dt Dt = ln 2 µ n log( ) n0 PD = ∏ log 2 Animal Cell Metabolism Metabolism • • • • Sources of carbon and energy: glucose and glutamine (and anabolic precursors) By-product metabolites: lactate and ammonia Source: Ratledge, C, Kristiansen, B, Basic Biotechnology, Cambridge University Press, 2001 Nutrient Metabolism •Limited supply of glucose can be compensated by an increased consumption of glutamine and viceversa “Flexibility” ” of animal cells Glutamine is also a nitrogen source for animal cells, so its limitation can lead to an increased consumption of other amino acids to compensate lower nitrogen intake. Metabolic Pathways Glucose Metabolism: Source: www.uic.edu/classes It is mainly metabolized via the glycolytic route to pyruvate; Metabolic Pathways Glucose Metabolism: Pyruvate is mainly reduced to lactate; Source: www.uic.edu/classes Lactate is excreted and accumulates in the culture medium; Metabolic Pathways Glucose Metabolism: Source: www.uic.edu/classes Alternatively, pyruvate may be converted to acetyl-CoA which enters the tricarboxylic acid cycle (TCA cycle); . Source: www.uic.edu/classes Metabolic Pathways Source: Wikipedia Metabolic Pathways Metabolic Pathways Glucose Metabolism: 4 – 8 % of consumed glucose goes via the pentose phosphate pathway (PPP) which supplies ribose-5-phosphate (R-5-P) for the synthesis of nucleotides, as well as reducing equivalents (NADPH) for biosynthesis. Metabolic Pathways Glutamine/Glutamate Metabolism: Metabolic Pathways Glutamine Metabolism: It can be catabolized in a number of ways, all beginning with deamination of glutamine that yields glutamate and NH4+; A smaller amount is metabolized via transamidation reactions in which the amide group is transferred to a precursor metabolite which is used in biosynthesis of purines, pyrimidines and aminosugar moities incorporated into glycan structures; The second step is the conversion of glutamate to α-ketoglutarate via glutamate dehydrogenase yielding another NH4+ via transamination with pyruvate or oxaloacetate as amino-group acceptors yielding alanine or aspartate. Alanine, in particular, is easily excreted from the cell and accumulates in the culture medium. Both glucose-derived and glutamine-derived carbon enter the TCA cycle, as acetyl-CoA and α-ketoglutarate, respectively. Inhibition by Metabolic By-products Lactate (20-60 mM) Inhibition of growth by the decrease in pH that follows from lactate excretion to the culture medium. Metabolic By-Products NH3 / NH4+ (1-5 mM) Inhibition includes interference with electrochemical gradients, changes in intracellular pH, apoptosis and a futile cycle of NH3 / NH4+ which increases the demand for maintenance energy. Also disturbs the glycosylation pattern of the product. Alanine (not believed to harm the cells) Cell Characterization in Tissue Culture Detection of Biochemical Components Many biochemical assays exploit the use of highly specific antibodies which are combined with enzymatic or fluorescent labels: o Enzymatic staining: color change in a substrate and production of immunohistochemical stains; o Fluorescent staining: immunofluorescent labeling Non-antibody-based techniques: • Expression of reporter fluorescent proteins: gene for the protein (e.g. GFP) is transferred to a cell and expressed continuoulsy with a protein of interest; the fusion protein can be tracked dynamically in live cells; • Expression of b-lactamase: genetic transfer of the gene to the cells; the enzyme cleaves a fluorescent substrate and produces a color change in proportion to the gene expression; In vivo Imaging Noninvasive: ultrasound, planar x-rays, fluoroscopy, computer-assisted tumoghraphy, magnetic resonance imaging and nuclear medicine scans, which uses radioisotipes to image organ function; Invasive: miniaturized fiberoptic probes which can be guided through a small incision or through a vessel. Measurement of Cell Characteristics Cell Morphology Morphology: form and structure of cells Qualitative assessment of the purity, general health and density of cell cultures; Cells can be described as being: • Fibroblastic (elongated and branched) • Epithelial-like (rounded and cobblestonelike patterns) • Lymphoblast-like (rounded and in suspension) Influenced by the density (confluence) of the culture; Unhealthy cells may show an increase in cytoplasmic granules (dark) and vacuoles (clear) or detach from the substrate. Cell number Hemocytometer: chamber of fixed volume with grid lines that can be observed under the microscope to count cells in suspension (also adherent cells after harvesting) Source: www.wisc.edu Source: www.uvm.edu Cell Counting Source: Domingos Henrique, Cell and Tissue Engineering Classes, IST, 2008-2009 Measurement of Cell Characteristics Cell number (cont.) Coulter counter: electronic particle counter for single-cell suspension Cell aggregation can limit the accuracy of this technique. Indirect methods: require to sacrifice the culture in order to measure the number of cells (so-called destructive assays): • DNA content • Protein content • Metabolic activity (ex. MTT, MTS, …) Require calibration to convert DNA or protein content, or metabolic activity to cell number. Measurement of Cell Characteristics Cell Viability Viability Criteria Methods Ability to attach and divide upon plating at very low density Clonogenic Assays Plasma-membrane integrity Trypan blue stain Polar nuclear stain Fluorescent cytoplasmic stains Lactate dehydrogenase release Metabolic activity Mitochondrial potential stain Presence of DNA Nonpolar nuclear stain Adapted from: Palsson, B.Ø. and Bhatia, S.N., Tissue Engineering, Pearson Prentice Hall Bioengineering, 2004 Viable cell fraction is assessed by light or fluorescent microscopy or flow cytometry analysis. Measurement of Cell Characteristics Cell Function: metabolism, protein synthesis, signal transduction… Function Studied Method Protein synthesis and secretion Incorporation of 3H-leucine, 35S-methionine and 3H-proline Protein-specific assays (ELISA) DNA synthesis Incorporation of 3H-thymidine, 3H-deoxycytidine and BrdU Metabolic activity Rate of consumption of glucose/glutamine/aminoacids/oxygen and rate of production of lactate/ammonia Signal transduction Intracellular calcium or pH dyes Microphysiometer (changes in extracellular pH) Biochemical assays on cell extracts Adapted from: Palsson, B.Ø. and Bhatia, S.N., Tissue Engineering, Pearson Prentice Hall Bioengineering, 2004 Measurement of Tissue Characteristics Tissue Properties: combination of both cells and extracellular matrix (ECM) General appearance: size, shape, color, weight…(microscopy, enzymatic tissue disruption…); Cellular component: results from a dynamic equilibrium between various cellular-fate processes (cell death, cell migration); ECM component: also a result of a dynamic equilibrium process (microscopy, radioisotope labeling, immunostaining…); Function: (ex. Contractility in cardiac tissue, insulin-producing function by β-islet cells in pancreas) Mechanical measurements: viscoelasticity, contractility, mechanical loading,… Physical properties: optical, acoustic, conductive, thermal… Cryopreservation Cryopreservation: storage of the cells by freezing Why? the ability to recover a well-characterized cell state for tissue-engineered medical products; the prevention of genotypic drift due to genetic instability; storage prior to senescence; prevention of transformation; control of phenotypic instability; a back-up in case of contamination; storage for characterization and pathogen testing. Cryopreservation Cryopreservation (cont.) How? Cells are usually removed from the substrate by enzymatic or mechanical disruption and suspended in a solution (typically, culture medium) that contains a cryoprotectant. Cryoprotectant: chemical additive such as dimethyl sulfoxide (DMSO) or glycerol that is used to prevent ice formation inside the cell, which is a major cause of cell damage. Cells are cooled and stored in a liquid nitrogen tank at -196ºC; Cooling rate is a critical parameter that affects subsequent cell survival (e.g. -1ºC per minute) Contaminants Contamination: can be suspected by a macroscopic change in the culture (cloudiness, observable colonies, change in pH…) 5 major types of microbes Bacteria: distinguished by their shape (rods, cocci or spheres) and observable motility; Yeast: round, refractile particles that increase in number over time; Fungi: observed as a combination of thin filamentous extensions and clusters of spores; Mycoplasma and viral detection: particularly difficult to observe; mycoplasma and some viruses pass through the sterile filters used for cell culture research (0.2 µm pore diameter); unexpected behavior of cultured cells: morphology, growth rate…; confirmed by specific assays (e.g. PCR). Prevention by using sterile culture techniques and antibiotics