1. Chapter 3
Biology of cultured cells
“The validity of the cultured cell as a model of physiological function in 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. Criticism: Culture Environment (II)
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, and proliferation of Unspecialized progenitor cells [well-differentiated cells ??]

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

5. Cell Adhesion (II) Glass: a slight net negative charge
Plastics (polystyrene): treated with an electron ion discharge or high-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. Two components of Interaction:
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
Anchoring junction
Tight junctions
Gap junctions
Extracellular matrix
Space between cells
Plasma membranes of adjacent cells
(desmosomes)

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

9. “between secretory cells” & “between endothelial cells”
Intercellular Junctions (II)
Anchoring junction
Tight junctions
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
Gap junctions
“between cardiomyocytes”
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 ECM Primary 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:

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. Dedifferentiation VS Differentiation
“Given the correct the culture conditions, differentiated functions can be reexpressed”

21. EDTA: sequester Ca2+, which is essential for the binding of annexin-V
Cell apoptosis assay
1. Annexin-V assessment:
EDTA: sequester Ca2+, which is essential 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 to substrate will form the basis of primary culture
Cells that are capable of proliferation will increase, some cell types will survive 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 renew in 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 stress in vivo --e.g., fibroblasts, muscle, glia --with a limited life span
Cells are cultured from a neoplasm