BIOLOGY OF CULTURED CELLS

THE CULTURE ENVIRONMENT

CELL ADHESION CAM cytoskeleton

CELL PROLIFERATIONCell cycle

DIFFERENTIATIONMaintenance of differentiationDedifferentiation

EVOLUTION OF CELL LINESsenescence

THE DEVELOPMENT OF CONTINUOUS CELL LINES

ORIGIN OF CULTURED CELLS

TRANSFORMATIONProperties of transformed cells

THE CULTURE ENVIRONMENT

Environment in vivo and in vitro can never be the sameIn vitro condition promote :• Spreading• Migration• Proliferation (of unspecialized cell)

Cells(invitro)Organ

(invivo)3D

Cells

-reduced cell-cell and cell-matrix interactions-lack heterogeneity -lack 3D architecture found in vivo-Hormonal & nutritional environment altered

5 ways - Influence of the environment on the culture :

i) Nature of substrate, growth phase (monolayer, semisolid, suspension)

ii) Medium (physicochemical and physiological constitution)

iii) Constitution of the gas phase

iv) Temperature

v) Degree of contact with other cells

Requirement for expression specialized function are the appropriate environment (nature, medium,substrate adhesion, nutrient, hormones and cell interactions etc )

CELL ADHESION

Normal cell from tissues grow as monolayers.Need to attach and spreadout on the substrate before they start to proliferate

Unless transformed cells-do not need to attach and is anchorage independent

Cells attach to substrate via secretion of extracellular matrix before spreading.

Examples of extracellular matrix: fibronectin, collagen or gelatin

These are commonly treated on substrates

CELL ADHESION…continue

Association: cell-cell and cell-substrate

- Mediated by cell surface glycoproteins and Ca2+- Proteoglycans (derived from cells and serum)- Cells also secrete the extracellular matrix and proteoglycans.

therefore;During subculture/passage of cells : Requires chelation of Ca2+ (Use EDTA) Degradation of extracellular matrix and degradation of cell adhesion molecules (use trypsin)

CELL ADHESION MOLECULES (CAM)

3 major classes of transmembrane proteins

Cell-cell interaction

(i) CAMs (Ca2+ independent)(ii) Cadherin (Ca2+ dependent)

Cell-matrix interaction

Via receptor for extracellular matrix : Integrins; receptors on the cells forMatrix molecule ie. Fibronection, Entactin, Laminin, Collagen(All bind through the RGD motive arg-gly-asp of the matrix molecules)

(iii) Transmembrane proteoglycan- interact also with matrix constituents Dissagregation of tissues by protease digest extracellular matrix Some cell more resistant e.g. intestinal ephitelial (tight junction complexes)

CAM attach to intracellular cytoskeleton (actin microfilaments, microtubules)Role related to cell motility, intracellular trafficking of micro-organelles or chromatids at cell division

ORIGIN OF CULTURED CELL

Original culture may contain an equilibrium of :

-Multipotent stem cell (precursor cells that can give rise to diverse cell types in response to appropriate environmental responds/signals-Undifferentiated but committed precursors-Mature differentiated cell

The equilibrium may shift according to the environmental conditions.

Routine serial passage:At low density, promote proliferation but constraint differentiationAt high density, low serum plus hormones promote differentiation and inhibit proliferation

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Culture source will determined which cellular components may be present:

- Embryo-contain more stem cells and precursor cell and be capable of greater self-renewal in vivo compared to adult

-Tissues continuously renewing ie. epithelium, haemopoietic cells- contain stem cells and prolong life span.

- Tissues that solely renew under stress ie. Fibroblast, glia – contain committed precursors cells with limited life span.

Two factors determined the identity of a culture cells:The lineage in vivo (hemopoietic, hepatocyte, glia)The position in the lineage (stem, committed precursor cell,

mature differentiated)

ORIGIN OF CULTURED CELL..continue

Two factors determined the identity of a culture cells:1.The lineage in vivo (haemopoietic, hepatocyte, glia)2.The position in the lineage (stem, committed precursor cell, mature differentiated)

CELL PROLIFERATION

Cell Cycle Control

The entry of cells into cycle is regulated by signals from the environment.

Cell ProliferationCell cycle is made up of Four phases :M : mitosis, chromatin condenses into chromosomes and chromatids that make up the chromosomes segregates to each daughter cell

G1 (gap 1) : checkpoint that control cell progress towards DNA synthesis or remain at G0 or commits to differentiation

S phase : DNA synthesis in which DNA replicates

G2 (Gap 2) : cell prepare for re-entry into mitosis

EVOLUTION OF CELL (PHENOPTYPIC DRIFT)

First subculture (or passage) – cell line that requires propagation

Successive subculture-Proliferative cell predominate, slow growth cell will diluted out-Selection for cell resistant to trauma of trypsinization / dissagregation / transfer

Third passage is more stable culture (of rapidly proliferating cell)

SenescenceNormal cell can divide a limited number of timeGenetically determined eventLinked to shortening of telomere, terminal sequences in DNA telomere not replicate.(except germ, stem or transformed cells)

Mitosis in cells

Lag phase : following seedingLog phase: exponential growth

Cell density (cells/cm2 of substrate(cell concentration: cells/ml medium

Fully occupied/exceed capacity

Change mediumDivide culture

SubcultureRemove medium

Dissociate cells (trypsinised)

Dilute in fresh mediumCell concentration usually 104/105 cells/ml

New bottles

DIFFERENTIATION

-The process cells undergo as they mature into normal cells. -Process leading to expression of phenotypic properties characteristic of the functionally matured cell in vivo.

Differentiated cells have distinctive characteristic -perform specific function and -less likely to divide.

Can be an irreversible process (cessation of DNA synthesis by erythrocyte) and can be reversible (eg. induction of albumin synthesis by hepatocytes-lost under certain culture conditions but may be re-induced)

DIFFERENTIATION .. continue

Differentiation can be constitutive or adaptive in mature cell.

Two ways of differentiation :' Commitment ' – irreversible transition from stem cell to particular lineage endowing cell with some properties (constitutive or induced)

`terminal differentiation’’cell had progressed to lineage to a point which the mature phenotype is fully expressed and beyond which cell cannot progresse.g neurons, skeletal muscle, keratinised squames.(differentiation is irreversible)

DEDIFFERENTIATION

Definition: `irreversible loss of the specialised properties that a cell would have expressed in vivo’

Or `the loss of differentiated properties of tissue when it becomes malignant or growth in culture (A mature cell returning to a less mature state). Loss of the phenotypic characteristics typical of the tissue from which the cells had been isolated (original)

Process reversal to differentiation: due to overgrowth of undifferentiated cells of the same or a different lineage

Need to provide correct conditions so that many of the differentiated properties of these cells may be restored

Serum-free selective media –allowed for the isolation of specific lineages

Contributory factors for dedifferentiation :

Cell deathSelective overgrowthAdaptive responseLoss of certain phenotypic properties

Dedifferentiation occurs through

i) adaptive processdifferentiation regain by giving the right inducer

ii) selective process

progenitor/precursor cells has been selected because of greater proliferative potential Stage of commitment and differentiation

DEVELOPMENT OF CONTINUOUS CELL LINES

Cell lines may be propagated in an unaltered form to a limited number of cell generations. Beyond that they may-die out

The ability of cell line to grow continuously probably reflects its capacity for genetic variation allowing subsequent selection.Some are diploid – never give rise to continuous cell linese.g. fribroblast, endothelial cell

Some are aneuploid – variety of human and animal tumour and give rise to continuous cell line.

The alteration in a culture to become a continuous cell line is called in vitro transformation.

How does it occur ?•Spontaneous•Chemically induced•Hormonally induced•Virally inducedTransformation-an alteration in growth characteristics•Anchorage independence•Loss of contact inhibition•Density limitation of growth•Continuous cell line are usually – aneuploid•(chromosome number between diploid and tetraploid value)

TRANSFORMATION is associated with three major classes of phenotypic change :

1) ImmortalizationTha acquisition of an infinite life span, presumed to be due to the deletion or mutation of one or more genes coding for senescence.Overexpression or mutation of one or more oncogenes that override the action of the senescence genes.Immortalization per se, does not imply either failure in growth control, or the development of malignancy.

2) Aberrant growth controlMay arise simultaneously with immortalization but can also occur as distinct subsequent event.Many cells progress readily from immortalization to aberrant growth, due perhaps to genetic instability intrinsic to the immortalized genotype.

3) MalignancyCell developed the capacity to generate invasive tumors if implanted in vivo into an isologous host.

Properties of transformed cells

Growth characteristics

- Immortal

- Anchorage independence: clone in agar, may grow in stirred suspension

- Loss of contact inhibition

- Growth on confluent monolayers of homologous cells “focus” formation

- Reduced density limitation of growth: high saturation density, high growth fraction at saturation density

- Low serum requirement

- Growth factor independent

- High plating efficiency

- Shorter population doubling time

Structural alteration Modified actin cytoskeleton

Loss of cell surface-associated fibronectin

Increased lectin agglutination

Modified extracellular matrix

Altered cell adhesion molecules

Disruption in polarity

Neoplastic properties Tumorigenic

Angiogenic

Enhanced protease secretion

Plasminogen activator

Invasive

Genetic properties

- High spontaneous mutation rate

- Aneuploid

- Heteroploid

- Overexpressed oncogenes

- Deleted suppressor genes

Normal human fibroblast-remain euploid throughout their life-span (~ 50 generations) will stop dividing.

Common feature of many human continuous cell lines is the development of subtetraploid chromosome number.