Biology of Cultured Cells Chapter 3. Influence of environment on culture - four routes The nature of the substrate on or in which cells grow The degree.

Biology of Cultured Cells

Chapter 3

Influence of environment on culture -four routes

The nature of the substrate on or in which cells grow

The degree of contact with other cells The physicochemical and physiological

constitution of the medium The constitution of the gas phase The incubation temperature

What factors allow for cell adhesion?

Attach and spread out on substrate before they proliferate

Grow in T-flasks or Petri dishes or Roux Bottles

Combination of electrostatic attraction and Van der Waal’s forces

Ca 2+ and basic proteins

Different Cell Adhesion Molecules

CAM’s

Cadherins

Integrins

Transmembrane proteoglycans

Cell-Cell Adhesion molecules

CAMs (Ca2+ independent) and Cadherins (Ca 2+ dependent)

Homologous cells interact

Cell-cell recognition generates signaling role in cell behavior

Cell-Substrate adhesion molecule

Integrins allow for cell-substrate interactions

Cell surface receptors for ECM (fibronectin, extractin, laminin and collagen)

Bind via RGD

Two polypeptide chains – α and β

Cell adhesion molecules

Transmembrane proteoglycans interact with matrix constituents such as proteoglycans or collagen

No RGD motif

What are intercellular junctions?

Cell adhesion molecules diffusely arranged in plasma membrane

Organized into intercellular junctions

Desmosomes, adherens, gap and tight junctions

What is extracellular matrix?

Intercellular spaces in tissues filled with ECM

Regulates phenotypic expression

Produced by cell cultures

Exogenous provision - experiments

How does protease act?

Digests some ECM and some extra cellular domains of transmembrane proteins

Epithelial and endothelial cells – resistant

Mesenchymal cells – less resistant

Confluency of epithelial cells

What elements of cytoskeleton are attached to cell adhesion molecules?

Integrins and Cadherins – actin cytoskeleton – in adherens junction

Cadherins – intermediate cytoskeleton – in intermediate junction – desmosomes

Microtubules

Do cells show motility?

Fibroblasts show directional migration Polar movement

Contact inhibition – directional migration ceases + reduction in plasma membrane ruffling

Leads to withdrawal of cell from division cycle

Eukaryotic Cell Cycle

The eukaryotic cell cycle has 5 main phases:

1. G1 (gap phase 1)2. S (synthesis)

3. G2 (gap phase 2)4. M (mitosis)5. C (cytokinesis)The length of a complete cell cycle varies greatly

among cell types.

interphase

Interphase

Interphase is composed of:

G1 (gap phase 1) – time of cell growth

S phase – synthesis of DNA (DNA replication)

- 2 sister chromatids are produced

G2 (gap phase 2) – chromosomes condense

Control of the cell cycle

The cell cycle is controlled at three checkpoints:

1. G1/S checkpoint-the cell “decides” to divide

2. S/G2 checkpoint - the cell “decides” DNA

repair or apoptosis

Control of the cell cycle

3. G2/M checkpoint

-the cell makes a commitment to mitosis

4. late metaphase (spindle) checkpoint

-the cell ensures that all chromosomes are attached to the spindle

How growth factors control the cell proliferation?

Growth factors:

- Epidermal growth factor (EGF), Fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF)

- Allows low cell density population to enter into cell cycle

- Does not allow proliferation of high cell density population

How cell cycle inhibitors control cell proliferation?

Rb gene product, p53, and p16

Block cell cycle progression/arrest

Damage leads to cancerous cells

Inactivation by phosphorylation-cell cycle progression

Factors that lead to Dedifferentiation

Inability of cell lines to differentiate- Wrong lineage of cells is selected in vitro

- Undifferentiated cells of same lineage overgrow terminally differentiated cells of reduced proliferative capacity

- Absence of inducers (hormones, cell or matrix interaction) can lead to reversible loss of differentiated properties

Different ways of cell-signaling

Signals reaching cells from another tissue via the systemic vasculature – Endocrine

Signals reaching cells from adjacent cells without entering bloodstream – Paracrine

Signals arising and interacting with same cell – Autocrine signaling

Different ways of cell-signaling

Signals arising and interacting with adjacent cells - Homotypic paracrine or homocrine signaling

Signals arising and interacting with different cells –Heterotypic paracrine

Types of cell-signaling in in vitro

Autocrine and homocrine signaling will occur

Slow growth due to dilution of autocrine or homocrine factors

Paracrine and endocrine factors are added

Energy metabolism

4-20mM glucose – carbon source for glycolysis

Glutamine – carbon source for citric acid cycle

- Deamination of glutamine produces NH3 - toxic and can limit cell growth

- Use of dipeptides such as glutamyl-alanine or glutamyl-glycine minimize production of NH3

Initiation and Evolution of Cell lines

Culture derived from main tissue – Primary Culture

Culture derived from primary culture – Cell Line

Continuous cultures or passage of cell cultures derived from cell line – Subculture

What is Senescence? Why and how does it happen?

Normal cell lines will die after fixed number of population doublings – Senescence

Inability of terminal sequences of DNA in telomeres to replicate at each cell division

Progressive shortening of telomeres – cell cannot divide

Why Senescence does not take place in all cells? Germ cells, stem cells, transformed cells, tumor

lines etc

Express enzyme telomerase which is capable of replicating the terminal sequences of DNA in telomeres

Extends life span of cells

What are Continuous Cell Lines?

Ability of cell lines to grow indefinitely in vitro

Alteration of a culture – TRANSFORMATION and giving rise to continuous cell line – IMMORTALIZATION

Shows capacity for genetic variation/instability

p53 is mutated or deleted (if tumor cell line)

How Continuous Cell Lines develop (Heteroploidy)? Aneuploidy –

chromosome number lies between diploid and tetraploid

Mouse fibroblasts and cell cultures from variety of human and animal tumors

Genetic instability is present

Human continuous lines develop tetraploidy

Origin of Culture

Cell lines derived from embryo are good for culturing than adult cells

Identity of cultured cell depends on two factors- Lineage of cell in vivo (hematopoietic, hepatocyte,

glial etc)- Position of cell in that lineage (stem cell,

precursor cell or mature differentiated cell)

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