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Chapter 3

Chapter 3. Biology of cultured cells. “The validity of the cultured cell as a model of physiological function in vivo has frequently been criticized ”. Culture Environment (I). The influence of the environment on the culture:.

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Chapter 3

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  1. Chapter 3 Biology of cultured cells “The validity of the cultured cell as a model of physiological functionin vivo has frequently been criticized”

  2. Culture Environment (I) The influence of the environment on the culture: • Nature of substrate which the cells grow: -- plastic, rigid matrix, collagen, agar, liquid • Degree of contact with other cells • Physicochemical and Physiological constitution of the medium • Constitution of gas phase • Incubation temperature

  3. Culture Environment (II) Criticism: • The cell does not express the correct in vivo phenotype • Cell-cell and Cell-matrix interactions are reduced • Many hormonal, neurotransmitters, and nutritional stimuli are absent • In vitro cell culture environment favors the spreading, migration, andproliferation of Unspecialized progenitor cells[well-differentiated cells ??]

  4. Cell Adhesion (I) “Cultured cells will need to attach and spread out on the substratebefore they will start to proliferate”

  5. Cell Adhesion (II) • Glass: a slight net negative charge • Plastics (polystyrene): treated with an electron ion discharge orhigh-energy ionizing radiation • Coating with matrix constituents: fibronectin, collagen, gelatin • Cell density:--fibroblast-like cells: seeding with LOW density--epithelial cells: seeding with HIGH density

  6. Cell Adhesion Molecules Two components of Interaction: (1) (Ca2+-indept.) (1) Cell-cell interaction (intercellular junction) (2) Cell-matrix interaction(extracellular matrix) (1) (Ca2+-dept.) (2) (2) (1) via RGD motif(Arg-Gly-Asp) NOT via RGD motif “Interaction between homologous cells; self-interactive”

  7. Intercellular Junctions Tight junctions Anchoring junction Gap junctions Extracellular matrix Space between cells Plasma membranes of adjacent cells (desmosomes)

  8. Intercellular Junctions (I) Tight junctions Anchoring junction Gap junctions Extracellular matrix Space between cells Plasma membranes of adjacent cells “between epithelial cells”

  9. Intercellular Junctions (II) Tight junctions Anchoring junction Gap junctions Extracellular matrix Space between cells Plasma membranes of adjacent cells “between secretory cells”&“between endothelial cells”

  10. Intercellular Junctions (III) Tight junctions Anchoring junction “between cardiomyocytes” Gap junctions Plasma membranes of adjacent cells

  11. Extracellular Matrix (ECM) • Fibroblasts: producing type I collagen, fibronectin, basal lamina • Epithelial cells: producing laminin • Epithelial keratinocuyes: basal lamina • Composition of the ECM regulates the cell phenotype • Cultured cell line: generating their own ECMPrimary cultured cell: requiring exogenous provision of ECM • Various ECM: collagen, fibronectin, laminin, hyaluronan, proteoglycans, growth factor, cytokines etc. • Preparation of ECM for cell culture:--mixing commercially purified constituents--using cell to generate EMC and washing the producer cells off--commercially available Matrigel (laminin+fibronectin+proteoglycans)[produced by Engelberth-Holm-Swarm mouse sarcoma]

  12. Disaggregation of an attached monolayer culture[using Protease + EDTA (Ca2+ chelating agent)] • Epithelial cells:--are more resistant to disaggregation (expressing tight junctions) • Endothelial cells:--are more resistant to dissociate (expressing tight junctions) • Mesenchymal cells:--are more easily dissociated--an undifferentiated cell found in mesenchyme and capable of differentiating into various cell types--all organs in the body contain mesenchyme(間葉組織) [=embryonic connective tissue] --capacity to differentiate into smooth muscle cells (in the formation new arteries), phagocytes (in inflammatory process) and bone cells (in the formation of new bone tissues)

  13. Signaling role of Cell-cell or Cell-matrix Interaction • Cell behavior:--”cell migration”-- “Contact Inhibition”  “Inhibition of proliferation” --”withdrawal” of the cell from the cell division cycle--”cell proliferation”--”cell apoptosis” • Integrins and Cadherins:-- interact with Vinculin (membrane-cytoskeletal protein)-- control cell’s motility-- signaling to the Nucleus

  14. Cell migration Endothelial cells Smooth muscle cells Chien-Cheng Hsieh & Ying-Tung Lau JOSEPH A. MADRI & KURT S. STENN, Eur J Physiol (1998) 435:286–292 Am J Pathol 1982, 106:180-186

  15. Cell Proliferation (I) Cell Cycle (1) (2)

  16. Flowcytometric analysis of DNA content DNA probes (like PI, propidium iodide) intercalates into cellular DNA. The fluorescent intensity indicates the number of chromosomes in each cell. Normal cells have diploid DNA content.

  17. Cell Proliferation (II) Extracellular Control:EGF, FGF, PDGF Intracellular Control:“Regulatory proteins” (1) Positive-acting factors: Cyclins (2) Negative-acting factors: p53, Rb

  18. Suppressor Genes Oncogenes Cancer Research

  19. Cell Proliferation (III) 1. MTT assay:yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) Intracellular reduction intracellular purple formazan (=570 nm) 2. [3H] Thymidine Incorporation: + 3H-Thymidine *

  20. DedifferentiationVSDifferentiation “Given the correct the culture conditions, differentiated functions can be reexpressed”

  21. Cell apoptosis assay 1. Annexin-V assessment: EDTA: sequester Ca2+, which isessential for the binding of annexin-V 2. DNA content assessment:

  22. A series of selective processes to a relatively uniform cell line Selection: • Only the cells that survive the disaggregation technique and adhere tosubstrate will form the basis of primary culture • Cells that are capable of proliferation will increase, some cell types willsurvive but not increase [the relative proportion of each cell type will change !!!]

  23. Evolution of a cell line In vitro transformation Senescence:“Progressive shortening of the telomers” [Exception: germ cells, stem cells, transformed cells (telomerase)]

  24. Continuous cell line: Aneuploid chromosome number Normal cell Continuous metastatic melanoma cell “Continuous cell line are usually aneuploid and often have a chromosome number between dipolid and tetraploid values”

  25. Origin of cultured cells • Cell line derived from the embryo (& adults)--Embryo may contain much more stem cells and precursor cells and be capable of greater self-renew • Cell line derived from the tissues undergoing continuous renewin vivo--e.g., epidermis, intestinal epithelium, hematopoietic cells--may contain stem cells & will have a prolonged life span • Cell line derived from the tissues that renew solely under stressin vivo--e.g., fibroblasts, muscle, glia--with a limited life span • Cells are cultured from a neoplasm

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