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

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:

1. Nature of substrate which the cells grow: -- plastic, rigid matrix, collagen, agar, liquid

2. Degree of contact with other cells

3. Physicochemical and Physiological constitution of the medium

4. Constitution of gas phase

5. Incubation temperature

Culture Environment (II)

Criticism:

1. The cell does not express the correct in vivo phenotype

2. Cell-cell and Cell-matrix interactions are reduced

3. Many hormonal, neurotransmitters, and nutritional stimuli are absent

4. In vitro cell culture environment favors the spreading, migration, andproliferation of Unspecialized progenitor cells[well-differentiated cells ??]

Cell Adhesion (I)

“Cultured cells will need to attach and spread out on the substrate

before they will start to proliferate”

Cell Adhesion (II)

1. Glass: a slight net negative charge

2. Plastics (polystyrene): treated with an electron ion discharge or high-energy ionizing radiation

3. Coating with matrix constituents: fibronectin, collagen, gelatin

4. Cell density:--fibroblast-like cells: seeding with LOW density--epithelial cells: seeding with HIGH density

(1) Cell-cell interaction (intercellular junction)

(2) Cell-m

atrix interactio

n(extracellu

lar matrix)

(1)

(1)

(2)(2)

(Ca2+-indept.)

(Ca2+-dept.)

(1)

“Interaction between homologous cells;

self-interactive”

via RGD motif(Arg-Gly-Asp) NOT via RGD motif

Two components of Interaction:Cell Adhesion Molecules

Anchoring junction

Tight junctions

Gap junctions

Extracellular matrix

Space between cells

Plasma membranes of adjacent cells

Intercellular Junctions

(desmosomes)

Anchoring junction

Tight junctions

Gap junctions

Extracellular matrix

Space between cells

Plasma membranes of adjacent cells

“between epithelial cells”

Intercellular Junctions (I)

Anchoring junction

Tight junctions

Gap junctions

Extracellular matrix

Space between cells

Plasma membranes of adjacent cells

Intercellular Junctions (II)

“between secretory cells”&

“between endothelial cells”

Anchoring junction

Tight junctions

Gap junctions

Plasma membranes of adjacent cells

Intercellular Junctions (III)

“between cardiomyocytes”

Extracellular Matrix (ECM)

1. Fibroblasts: producing type I collagen, fibronectin, basal lamina

2. Epithelial cells: producing laminin

3. Epithelial keratinocuyes: basal lamina

4. Composition of the ECM regulates the cell phenotype

5. Cultured cell line: generating their own ECMPrimary cultured cell: requiring exogenous provision of ECM

6. Various ECM: collagen, fibronectin, laminin, hyaluronan, proteoglycans, growth factor, cytokines etc.

7. 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]

Disaggregation of an attached monolayer culture[using Protease + EDTA (Ca2+ chelating agent)]

1. Epithelial cells:--are more resistant to disaggregation (expressing tight junctions)

2. Endothelial cells:--are more resistant to dissociate (expressing tight junctions)

3. 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)

Signaling role of Cell-cell or Cell-matrix Interaction

1. Cell behavior:--”cell migration”-- “Contact Inhibition” “Inhibition of proliferation” --”withdrawal” of the cell from the cell division cycle

--”cell proliferation”--”cell apoptosis”

2. Integrins and Cadherins:-- interact with Vinculin (membrane-cytoskeletal protein)-- control cell’s motility-- signaling to the Nucleus

Cell migration

Chien-Cheng Hsieh & Ying-Tung Lau

Eur J Physiol (1998) 435:286–292

Smooth muscle cellsEndothelial cells

JOSEPH A. MADRI & KURT S. STENN,

Am J Pathol 1982, 106:180-186

Cell Proliferation (I)

Cell Cycle

(1)

(2)

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.

Flowcytometric analysis of DNA content

Cell Proliferation (II)

(1) Positive-acting factors: Cyclins

(2) Negative-acting factors: p53, Rb

Extracellular Control:EGF, FGF, PDGF

Intracellular Control:“Regulatory proteins”

Suppressor Genes

Oncogenes

Cancer Research

Cell Proliferation (III)

1. MTT assay:yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)

intracellular purple formazan (=570 nm)

Intracellular reduction

2. [3H] Thymidine Incorporation:

+ 3H-Thymidine

*

Dedifferentiation VS Differentiation

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

Cell apoptosis assay

1. Annexin-V assessment:

EDTA: sequester Ca2+, which isessential for the binding of annexin-V

2. DNA content assessment:

A series of selective processes to a relatively uniform cell line

(1)Only the cells that survive the disaggregation technique and adhere tosubstrate will form the basis of primary culture

(2)Cells that are capable of proliferation will increase, some cell types willsurvive but not increase [the relative proportion of each cell type will change !!!]

Selection:

Evolution of a cell line

Senescence:“Progressive shortening of the telomers”[Exception: germ cells, stem cells, transformed cells (telomerase)]

In vitro transformation

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”

Origin of cultured cells

1. Cell line derived from the embryo (& adults)--Embryo may contain much more stem cells and precursor cells and be capable of greater self-renew

2. 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

3. Cell line derived from the tissues that renew solely under stress in vivo--e.g., fibroblasts, muscle, glia--with a limited life span

4. Cells are cultured from a neoplasm