plant and mammalian tissue culture mammalian cell culture

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  • Slide 1
  • Plant and Mammalian Tissue Culture Mammalian Cell Culture
  • Slide 2
  • Tissue Culture Tissue Culture: The general term for the removal of cells, tissues or organs from an animal or plant and their subsequent placement into an artificial environment conducive to growth. Can be used to prepare finite or continuous cell cultures Will be similar biochemically and physiologically to parent tissue
  • Slide 3
  • Organ Culture Organ Culture The culture of whole organs or intact organ fragments with the intent of studying their continued function or development. Can be maintained for hours or days by perfusion with oxygenated blood or serum. Used for metabolism and drug studies Provides most accurate reflection of organisms physiology
  • Slide 4
  • Organ Culture Organ Culture U of Minnesota 2007 heart perfusion w/stem cells created a new heart. (Click here for movie)Click here for movie
  • Slide 5
  • Cell Culture Cell Culture When cells are removed from the organ fragments prior to, or during cultivation, thus disrupting their normal relationships with neighboring cells. General term explaining non-in vivo experiments Non-tissue growth of plant and animal cells
  • Slide 6
  • Mammalian Cell Culture Mammalian Cell Culture Cell culture of mammalian cells. Eukaryotic cells are much more difficult to culture than most prokaryotes. They demand complex media They are very susceptible to contamination and overgrowth by microbes such as bacteria, yeasts and fungi.
  • Slide 7
  • Cell Culture Two types of cell culture Primary Culture Cell Line Culture AKA finite / continuous / established / secondary / subclone / immortalized cell culture
  • Slide 8
  • Primary Culture Come from the outgrowth of migrating cells from a piece of tissue or from tissue that is disaggregated by enzymatic, chemical, or mechanical methods. Formed from cells that survive the disaggregation process, attach to the cell culture vessel (or survive in suspension), and proliferate.
  • Slide 9
  • Primary Culture Primary cells are morphologically similar to the parent tissue. These cultures are capable of only a limited number of cell divisions, after which they enter a nonproliferative state called senescence and eventually die out.
  • Slide 10
  • Primary Culture Primary cells are considered by many researchers to be more physiologically similar to in vivo cells. Primary cell culture is generally more difficult than culture of continuous cell lines.
  • Slide 11
  • Primary Culture Advantages They are thought to represent the best experimental models for in vivo situations. Have the same karyotype as the parent tissue normal or abnormal. Not dedifferentiated Disadvantages Difficult to obtain. Relatively short life span in culture. Very susceptible to contamination May not fully act like tissue due to complexity of media Considerable variation in population and between preparations
  • Slide 12
  • Primary Culture Tumor Primary Cell Culture Easier to create as tumors cell cycle / growth regulators have been altered Tumor cells often produce own growth factors (autocrine) Mechanically disrupted tissue easily plates, binds and can thrive Seeding density often must be high for primary culture of tumors.
  • Slide 13
  • Finite Cell Lines AKA secondary or subclone culture Finite cell cultures are formed after the first subculturing (passaging) of a primary cell culture. These cultures will proliferate for a limited number of cell divisions, after which they will senesce. The factors which control the replication of such cells in vitro are related to the degree of differentiation of the cell
  • Slide 14
  • Finite Cell Lines The cells will proliferate for an extended time, but usually the culture will eventually cease dividing, similar to senescent primary cells. Use of such cells is sometimes easier than use of primary cell cultures, especially for generation of stably transfected clones.
  • Slide 15
  • Finite Cell Lines MRC5 cells Human embryonic lung fibroblasts Undergo between 60-70 doublings before senescence.
  • Slide 16
  • Finite Cell Lines Advantages Can obtain a large population of similar cells. Most cellular characteristics are maintained Can transform cells to grow indefinatly Disadvantages Cells have a tendency to differentiate over time in culture. Over time the culture tends to select for aberrant cell
  • Slide 17
  • Continuous Cell Line A cell line that has demonstrated the potential to be subcultured indefinitely. Infinite cell line Immortal cells line Immortalized cell lines are also known as transformed cells: Cells whose growth properties have been altered.
  • Slide 18
  • Continuous Cell Line Finite cell cultures will eventually either die out or acquire a stable, heritable mutation that gives rise to a continuous cell line that is capable of unlimited proliferative potential. This alteration is commonly known as in vitro transformation or immortalization and frequently correlates with tumorigenicity.
  • Slide 19
  • Continuous Cell Line Continuous cell lines are generally easier to work with than primary or finite cell cultures. These cells have undergone genetic alterations and their behavior in vitro may not represent the in vivo situation.
  • Slide 20
  • HeLa Cells Classic example of an immortalized cell line. These are human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18).
  • Slide 21
  • Continuous Cell Line Advantages Easy to maintain in culture. Easy to obtain large population of cells. Typically easy to manipulate gene expression. Disadvantages The more aggressive the cell line the more it changes over time in culture. Not clear how the function of these cells relates to that of other cells, healthy or diseased.
  • Slide 22
  • Transformed Cells Transformed, Infinite or Established Cells Changed from normal cells to cells with many of the properties of cancer cells. Some of these cell lines have actually been derived from tumors or are transformed spontaneously in culture by mutations. Chemical or gamma ray treated cells can become infinite with loss of growth factors Viral infection with SV40 T antigen can insert oncogenes and lead to p58 and RB gene alteration No matter how transformation occurred, the result is a cell with altered functional, morphological, and growth characteristics.
  • Slide 23
  • Know Your Cells The more you know about the cells and the more finely attuned you are to the cells quirks, the quicker and more clear the interpretation of results will be.
  • Slide 24
  • Know Your Cells The more differentiated the cell line, the slower it will grow. Categories of cell cultures based on origins. OriginSimilarity to original tissue Ease of maintenanceDoublings Primary cellsAnimal tissue, fetal or adult RepresentativeDifficult0 - 1 Finite cell linesAnimal tissue, usually fetal RepresentativeDifficultFetal: 20-80; adult tissue: very limited Continuous cell linesSpontaneous tranformation of primary or finite cell lines Not very representative; cell are less differentiated EasyIndefinite, with selection for higher growth rate Transformed cell linesTumor tissue, spontaneous or viral transformation of continuous cell line Not very representative; less differentiated than parent EasyIndefinite, with selection for higher growth rate Hybridoma (monoclonal antibody production) Fusion of antibody secreting B cells and myeloma cells Not representative of either cell type DifficultLimited
  • Slide 25
  • Know Your Cells Adherent or monolayer cells must bind to solid surface to survive and propagate. Suspension cells may need stirring or simply placed in flask.
  • Slide 26
  • Know Your Cells Wash these cells very carefully, as the loose monolayer can be inadvertently aspirated away. Adherently cultured transformed cells are usually highly anchorage-independent and adhere lightly even to tissue culture dishes.
  • Slide 27
  • Characterization by Cell Growth Attachment Cultures To survive and grow, most cells require a surface to which they can attach Without the surface attachment these cells cannot survive Anchorage-Independent Do not require attachment for cell proliferation Growth of cells in tissue culture dishes looks more haphazard than the growth of anchorage dependent cells with cells only loosely attached to the surface.
  • Slide 28
  • Characterization by Cell Growth The advantages of adherent growth is the ability of the cells to adhere and spread on surfaces such as coverslips, making microscopy, hydribidizations, and functional assays more easily performed.
  • Slide 29
  • Characterization by Cell Growth Suspension Cultures Some cells can survive and divide while being suspended in a fluid media and stirred or shaken. Flasks Spinner Cultures Shaker Cultures A limited number of cell types can be maintained and grown in either format.
  • Slide 30
  • Characterization by Cell Growth The advantages of suspension growth are the large numbers of cells that can be achieved, and the ease of harvesting.
  • Slide 31
  • Growing Cells in Culture Place cells

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