Mammalian Cell Culture

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Biology of cultured cells
Dr. Tarek Elbashiti Assoc. Prof. of Biotechnology
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• In vitro cell often does not express the correct
  in vivo phenotype because the cells
  microenvironment has changed.
• Cellcell and cellmatrix interactions are
  reduced because the cells lack the heterogeneity
  and three-dimensional architecture found in vivo,
  and many hormonal and nutritional stimuli are
  absent.

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• The influence of the environment on the culture
  is expressed via five routes
• (1) the nature of the substrate on or in which
  the cells growsolid, as on plastic or other
  rigid matrix, semisolid, as in a gel such as
  collagen or agar, or liquid, as in a suspension
  culture
• (2) the degree of contact with other cells
• (3) the physicochemical and physiological
  constitution of the medium
• (4) the constitution of the gas phase
• (5) the incubation temperature.

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• The providing of the appropriate environment,
  including substrate adhesion, nutrient and
  hormone or growth factor concentration, and cell
  interaction, is fundamental to the expression of
  specialized functions

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Cell adhesion

• Most cells from solid tissues grow as adherent
  monolayers, and, unless they have transformed
  and,
• Become anchorage independent after tissue
  disaggregation or subculture they will need to
  attach and spread out on the substrate before
  they will start to proliferate.

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• Cells attach to and spread on glass that had a
  slight net negative charge.
• Attach to some plastics, such as polystyrene, if
  treated with an electric ion discharge or
  high-energy ionizing radiation.
• Cell adhesion is mediated by specific cell
  surface receptors for molecules in the
  extracellular matrix so it seems likely that
  spreading may be preceded by the secretion of
  extracellular matrix proteins and proteoglycans
  by the cells.

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Cell Adhesion Molecules

• Three major classes of transmembrane proteins
  have shown to be involved in cell-cell and
  cell-substrate adhesion
• Cell-cell adhesion molecules, CAMs (Ca2
  independent), and cadherins (Ca2 dependent) for
  interactions between homologous cells.
• Cell-substrate interactions by integrins,
  receptors for matrix molecules such as
  fibronectin, laminin, and collagen.
• Transmembrane proteoglycans, also interacting
  with matrix such as other proteoglycans or
  collagen

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CELL JUNCTIONS

• Adherens junctions
• Gap junctions
• Tight junctions
• Desmosomes/Hemidesmosomes
• Focal adhesions

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Intercellular Junctions

• The role of the junctions varies between
  mechanical, such as the desmosomes and adherens
  junctions, which hold epithelial cells together
• Tight junctions which seal the space between
  cells, e.g. between secretory cells in ducts or
  between endothelial cells in a blood vessel, and
• Gap junctions, which allow ions, nutrients, and
  small signaling molecules such as cyclic
  adenosine monophosphate (cAMP) to pass between
  cells in contact.

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• Desmosomes distributed throughout the area of
  plasma membranes in contact they are often
  associated with tight and adherens junctions.
• Desmosomes are molecular complexes of cell
  adhesion proteins and linking proteins that
  attach the cell surface adhesion proteins to
  intracellular keratin cytoskeletal filaments.

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• As epithelial cells differentiate in confluent
  cultures they can form an increasing number of
  desmosomes and, if some morphological
  organization occurs, can form complete junctional
  complexes.

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• This is one reason why epithelial cells, if left
  at confluence for too long, can be difficult to
  disaggregate.
• As many of the adhesion molecules within these
  junctions depend on Ca2 ions, a chelating agent,
  such as EDTA, is often added to the trypsin
  during or before disaggregation.

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Extracellular Matrix

• Intercellular spaces in tissues are filled with
  extracellular matrix (ECM), the constitution of
  which is determined by the cell type, e.g.,
  fibrocytes secrete type I collagen and
  fibronectin into the matrix,
• Epithelial cells produce laminin.
• Where adjacent cell types are different, e.g., at
  the boundary of the dermis (fibrocytes) and
  epidermis (epithelial keratinocytes), both cell
  types contribute to the composition of the ECM,
  often producing a basal lamina.

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• The matrix adheres to the charged substrate, and
  the cells then bind to the matrix via specific
  receptors.
• Glass or plastic that has been conditioned by
  previous cell growth can often provide a better
  surface for attachment,
• Substrates pretreated with matrix constituents,
  such as fibronectin or collagen, or derivatives,
  such as gelatin, help fastidious cells to attach
  and proliferate.

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• Mostly, cultured cell lines are allowed to
  generate their own ECM,
• but primary culture and propagation of some
  specialized cells, and the induction of their
  differentiation, may require exogenous condition
  of ECM.
• ECM is comprised variously of collagen, laminin,
  fibronectin, hyaluronan, proteoglycans, and bound
  growth factors or cytokines

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• With fibroblast-like cells, the main requirement
  is for substrate attachment and spreading and the
  cells migrate individually at low densities.
• Epithelial cells may also require cellcell
  adhesion for optimum survival and growth and, as
  a result, they tend to grow in patches.

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• At least two components of interaction with the
  substrate may be recognized
• (1) Adhesion, to allow the attachment and
  spreading that are necessary for cell
  proliferation.
• (2) Specific interactions, such as of the
  interaction of an epithelial cell with basement
  membrane (a dense layer of extracellular
  material) with other ECM constituents, or with
  adjacent tissue cells, and required for the
  expression of some specialized functions explored
  the growth of cells on other natural substrates
  related to basement membrane.

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• The use of ECM constituents can be highly
  beneficial in enhancing cell survival,
  proliferation, or differentiation
• Fibronectin and laminin fragments are now
  available commercially

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CELL-CELL ADHESION MOLECULES

• Cadherins
• Ig superfamily CAMs
• Selectins
• Integrins
• Connexins (Gap Junction molecules)
• Occludin and claudin proteins

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CELL-CELL ADHESION MOLECULES

• Transmembrane proteins involved in cellcell and
  cellsubstrate adhesion
• 1- Cellcell adhesion molecules, CAMs
  (Ca2independent), by means of cell adhesion
  molecules, CAMs, cells are capable of recognizing
  each other
• Plasma membrane receptors take care of cell-ECM
  interactions
• 2- cadherins (Ca2 dependent) primarily involved
  in interactions between homologous cells

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• Cellsubstrate interactions are mediated
  primarily by integrins, receptors for matrix
  molecules such as fibronectin, entactin, laminin,
  and collagen, which bind to them via a specific
  motif usually containing the arginineglycineaspa
  rtic acid (RGD) sequence
• Each integrin comprises one a and one ß subunit,
  the extracellular domains of which are highly
  polymorphic, thus generating considerable
  diversity among the integrins.

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• The third group of cell adhesion molecules is the
  transmembrane proteoglycans, also interacting
  with matrix constituents such as other
  proteoglycans or collagen, but not via the RGD
  motif.
• Disaggregation of the tissue, or an attached
  monolayer culture, with protease digest some of
  the extracellular matrix and may even degrade
  some of the extracellular domains of
  transmembrane proteins, allowing cells to become
  dissociated from each other.

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• Epithelial cells are generally more resistant to
  disaggregation, as they tend to have tighter
  junctional complexes (desmosomes, adherens
  junctions, and tight junctions) holding them
  together, whereas mesenchymal cells, which are
  more dependent on matrix interactions for
  intercellular bonding, are more easily
  dissociated.
• Endothelial cells (specialized type of epithelial
  cell which forms the inner layer of blood
  vessels) may also express tight junctions in
  culture, especially if left at confluence for
  prolonged periods on a preformed matrix, and can
  be difficult to dissociate.

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• C.T Functions to bind and support other tissues
• Made up of a thin population of cells scattered
  through an extracellular matrix
• Matrix nonliving, web of fibers embedded in a
  homogenous ground substance that may be liquid,
  solid, or jelly-like
• Substances of the matrix are secreted by cells
  of connective tissues
• In each case, cells must re-synthesize matrix
  proteins before they attach or must be provided
  with a matrix-coated substrate.

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The binding is homophilic

• Adhesion may be homophilic (one cadherin binds to
  another in the extracellular space and connects
  cells together at specialized junctions) or
  heterophilic (binding protein binds to another
  type of site on a cell

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Cadherins

• Cadherins Large family of cell surface proteins
  that mediate homophilic Ca dependent cell-cell
  adhesion.
• The classical cadherins, e.g. E-,N-, P-,
  L-cadherins occur in the epithelial, neuronal
  placental and liver tissues respectively.

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• 700-750 a.a glycoprotein
• Five fold external parts
• Four with Ca
• In Ca deficiency !!
• Ca2 causes dimerization of Cadherins

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Cell-cell Adhesion Regulation of cadherins
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CADHERINS

• They have a critical role in embryo
  morphogenesis.
• In adult, cadherins are responsible for the tight
  cell-cell associations within tissues.
• They are closely associated with the
  cytoskeleton (actin ), By 3 intracellular
  proteins ( catenins)
• Associated with signaling between the cell
  surface and the nucleus
• If catenins absent cadherin dont act.

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CADHERINS

• Experiment (specifications)
• Fibroblast cell called L- fibroblast doesn't have
  cadherins , if transfected with gene encoding E
  cadherins , then use it.
• Without Ca disrupted
• Cells express multiple cadherins- specificity of
  adhesion is due to the different combinations of
  cadherins expressed.

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Cadherin-dependent Cell Sorting
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SELECTINS

• Surface carbohydrate binding protein.
• Ex. Lectins in the presence of Ca ions bind to
  specific oligosaccharides on another cell
• All are structurally closely related having, at
  their N-terminal a carbohydrate recognition (
  lectin) domain and variable numbers of repeats
  related to complement regulatory proteins.
• Heterophelic cell adhesion bind protein to
  another type on a cell)
• This binding is weak untill bind tightly by their
  integrins

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SELECTINS

• There are three types of selectins
• E-selectin, found exclusively on endothelia
• L-selectin, found on all circulating leukocytes
  except activated T-lymphocytes
• P-selectin, found in secretory granules of
  platelets and endothelial cells.

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Selectin-mediated cell-cell adhesion (blood cell
rolling)
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SELECTINS

• Selectins are involved in extravasations
• Inflammatory signals activate endothelial cells
  making P-Selectin undergo exocytosis
• P-Selectin on the surface of endothelial cells
  binds a specific carbohydrate ligand on
  leukocytes
• The leukocytes attach to the endothelial wall and
  roll slowly on it
• Platelet-activating factor (PAF) and integrins
  are then activated and the leukocytes start to
  pass through the walls of a vessel into the
  surrounding tissues.

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Integrins

• Transmembrane binding glycoproteins that usually
  bind cells to matrix
• Bind cells to cells and to specific legand on the
  target cell
• Involved in cell-extracellular matrix adhesion
  and cell-cell adhesion
• Binding is Ca dependent
• May involve actin filaments but not associated
  with cells junctions
• Functional integrins always have ß and a subunits

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• Ligand binding is divalent cation dependent
• (Ca , Mg and Mn)
• Common ligands are
• the ECM proteins ( fibronectin, vitronectin,
  collagen and laminin -recognised by multiple
  integrins-or members of the Ig superfamily)

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• Integrins connect the actin cytoskeleton to
  extracellular matrix proteins outside the cell.
• The clustering of integrins form a central
  adhesions facility.

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Many cells in culture do not proliferate in
response to growth factors unless the cells are
attached via integrins to extracellular matrix
molecules. The challenge is to determine how
these signaling cascades interact to influence
complex cell behaviors such as gene expression
and cell proliferation
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Ca independent cell-cell adhesion molecule

• Ig-superfamily of adhesion molecules
• includes around 70 members.
• All posses one or more Ig-like domain.
• Ig-like domains are - ß sheets proteins
  stabilized by disulphide bond.
• Ig domains are resistant to proteases

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Fig (B) NCAM recognising another NCAM molecules
on a different cell (homophilic ligand).
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• They recognise both homophilic and heterophilic
  ligands.
• Integrins are frequently heterophilic ligands
  for Ig-superfamily members
• e.g. ICAM binds to ß 2-integrins on blood cells.
• Ca dependency for ligand binding is variable.

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Neural cell adhesion molecule (N-CAM),
intercellular adhesion molecule
(ICAM) Calcium-independent Immunoglobulin (Ig)
superfamily
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Summary Diagramnon-junctional adhesion proceeds
junctional adhesion
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Cell Matrix Interaction
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• The extracellular matrix, where most animal cells
  in tissues are embedded, fills the spaces between
  cells and binds cells and tissues together.
• The wide matrix in CT may be calcified,
  transparent, liquid,
• Matrix form basal lamina between CT and
  Epithelial cells.

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• Basal laminae is a sheetlike extracellular matrix
  that supports epithelial cells and surrounds
  muscle cells, adipose cells, and peripheral
  nerves.

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• Matrix play more active and complex role in
  regulation the behavior of cells that contact it.
• Proliferation , migration, development, shape and
  function.
• Extracellular matrix is made and oriented by
  cells within it.
• Extracellular matrices are composed of
• 1- A gel-like polysaccharide ground substancea
  design basically similar to that of plant cell
  walls.
• 2-Tough fibrous proteins .

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1- Glycosaminoglycans GAGs

• GAGs,are gel-forming polysaccharides of the
  extracellular matrix that consist of repeating
  units of disaccharides.
• Five major types, have Differences in
• (sulphate group and its location, linkage type)

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GAGs
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GAGsprotein

• Proteoglycans are proteins linked to
  glycosaminoglycans and consist of up to 95
  polysaccharide by weight.
• E.g aggrecan
• A number of proteoglycans interact with
  hyaluronan to form large complexes in the
  extracellular matrix.

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GAGs function

• 1- GAGs act as filter gel
• Heparan sulphat proteoglycans golmerular basal
  lamina filter
• 2-Bind to growth factors
• 3- Regulation of secretory proteins by
• ( immobilize, delayed, prolong action )

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2-Tough fibrous proteins .

• Collagen, the major structural protein of the
  extracellular matrix, is the single most abundant
  protein in animal tissues.
• The collagens are a large family of proteins
  containing at least 27 different members

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-Stiff -Long -Triplehelix -With proline and
glycine -27 types -the famous isType 1 in CT.
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Elastin

• Elastic fibers, found in connective tissues, are
  particularly abundant in organs that regularly
  stretch and then return to their original shape.
• Elastin is cross-linked into a network by
  covalent bonds formed between the side chains of
  lysine residues and the protein that elastic
  fibers are principally composed of.

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Matrix adhesion proteins

• 1- Fibronectin is the principal adhesion protein
  of the connective tissues
• Is the final class of extracellular matrix
  constituents
• Responsible for linking the components of the
  matrix to one another and to the surfaces of
  cells.

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Fibronectin

• Large glycoprotein
• A dimer of two lage
• subunits linked by
• disulfide bond.

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2- Laminin

• Laminin is the principal adhesion protein of
  basal laminae.
• Entactin, another adhesion molecule that is
  associated with laminins, binds to type IV
  collagen.

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• BASAL LAMINA
• Flexible thin 40-120 nm
• Influence cell metabolism
• Induce cell differentiation from the cells which
  set on, two layer ( from collagen , perelecan
  heparan sulphate proteiglycan-, laminin and
  entactin )

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laminins
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Integrins

• Integrins are the major cell surface receptors
  responsible for the attachment of cells to the
  extracellular matrix.
• In addition to attaching cells to the
  extracellular matrix, integrins serve as anchors
  for the cytoskeleton.

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integrins

• Focal adhesions are a type of cell-matrix
  junction that attach a variety of cells,
  including fibroblasts, to the extracellular
  matrix.
• The ability of integrins to reversibly bind
  matrix components is dependent on their ability
  to change conformation between active and
  inactive states.

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Cell Motility

• Cultured cells are capable of movement on a
  substrate.
• The most motile are fibroblasts at a low cell
  density (when cells are not in contact), and the
  least motile are dense epithelial monolayers.
• Fibroblasts migrate as individual cells with a
  recognizable polarity of movement.
• A lamellipodiuma cytoskeletal protein
  actinprojection on the mobile edge of the cell ,
  generated by polymerization of actin expands in
  the direction of travel and adheres to the
  substrate, and the plasma membrane at the
  opposite side of the cell retracts, causing the
  cell to undergo directional movement.

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• If the cell encounters another cell, the polarity
  reverses, and migration proceeds in the opposite
  direction.
• Migration proceeds in irregular tracks, until the
  cell density reaches confluence, whereupon
  directional migration ceases.
• The cessation of movement at confluence, which is
  accompanied by a reduction in plasma membrane
  disturbing, is known as contact inhibition and
  leads eventually to withdrawal of the cell from
  the division cycle depending on the
  microenvironment.

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• Epithelial cells, unless transformed, tend not to
  display
• random migration as polarized single cells. When
  seeded at a low density, they will migrate until
  they make contact
• with another cell and the migration stops.
• Eventually, cells accumulate in patches and the
  whole patch may show signs of coordinated movement

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CELL PROLIFERATION Cell Cycle

• The cell cycle is made up of four phases
• In the M phase (M mitosis), the chromatin
  condenses into chromosomes, and the two
  individual chromatids, which make up the
  chromosome, separate to each daughter cell.
• In the G1 (Gap 1) phase, the cell either
  progresses toward DNA synthesis and another
  division cycle or exits the cell cycle reversibly
  (G0) or irreversibly to commit to
  differentiation.

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• G1is followed by the S phase (DNA synthesis), in
  which the DNA replicates.
• S in turn is followed by the G2 (Gap 2) phase in
  which the cell prepares for reentry into mitosis.
• Checkpoints at the beginning of DNA synthesis and
  in G2 determine the integrity of the DNA and will
  halt the cell cycle to allow DNA repair or entry
  into apoptosis if repair is impossible.

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Cell cycle regulation
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• Apoptosis, or programmed cell death is a
  regulated physiological process whereby a cell
  can be removed from a population.
• Marked by DNA fragmentation, nuclear blebbing,
  and cell shrinkage, apoptosis can also be
  detected by a number of marker enzymes with kits
  such as Apotag (Oncor) or the COMET assay
• Entry into the cell cycle is regulated by signals
  from the environment.

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Control of Cell Proliferation

• Low cell density leaves cells with free edges and
  renders them capable of spreading, which permits
  their entry into the cycle in the presence of
  mitogenic growth factors, such as epidermal
  growth factor (EGF), fibroblast growth factors
  (FGFs), or platelet-derived growth factor (PDGF)
  interacting with cell surface receptors.
• High cell density inhibits the proliferation of
  normal cells (though not transformed cells).

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• Inhibition of proliferation is initiated by cell
  contact and is highlighted by crowding and the
  resultant change in the shape of the cell and
  reduced spreading.
• Intracellular control is mediated by
  positive-acting factors, such as the cyclins ,
  which are upregulated by signal transduction
  cascades activated by phosphorylation of the
  intracellular domain of the receptor when it is
  bound to growth factor.

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• Much of the evidence for the existence of these
  steps in the control of cell proliferation has
  emerged from studies of oncogene and suppressor
  gene expression in tumor cells, with the ultimate
  objective of the therapeutic regulation of
  uncontrolled cell proliferation in cancer.
• The immediate benefit, however, has been a better
  understanding of the factors required to regulate
  cell proliferation in culture

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Dedifferentiation

• Historically, the inability of cell lines to
  express
• the characteristic in vivo phenotype was blamed
  on
• dedifferentiation.
• Dedifferentiation involves that the specialized
  properties of the cell are lost by change to a
  more primitive phenotype.
• According to this concept, differentiated cells
  lose their specialized properties in vitro, but
  it is often unclear whether (1) the wrong lineage
  of cells is selected in vitro, (2)
  undifferentiated cells of the same lineage
  overgrow terminally differentiated cells of
  reduced proliferative capacity,

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• or (3) the absence of the appropriate inducers
  (hormones cell or matrix interaction) causes an
  adaptive, and potentially reversible, loss of
  differentiated properties
• In practice, all of these may contribute to loss
  of differentiation even in the correct
  lineage-selective conditions

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CELL SIGNALING

• Cell proliferation, migration, differentiation,
  and apoptosis in vivo are regulated by cellcell
  interaction, cellmatrix interaction, and
  nutritional and hormonal signals, as discussed
  before.
• Some signaling is contact-mediated via cell
  adhesion molecules but signaling can also result
  from soluble, diffusible factors.
• Signals that reach the cell from another tissue
  via the systemic vasculature are called
  endocrine, and those that diffuse from adjacent
  cells without entering the bloodstream are called
  paracrine.

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• It is useful to recognize that some soluble
  signals arise in, and interact with the same type
  of cell.
• This called homotypic paracrine, or homocrine,
  signaling .
• Signals that arise in a cell type different from
  the responding cells are heterotypic paracrine
  and referred as paracrine.
• A cell can also generate its own signaling
  factors that bind to its own receptors, and this
  is called autocrine signaling.

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• Although all of these forms of signaling occur in
  vivo, under normal conditions with basal media in
  vitro, only autocrine and homocrine signaling
  will occur.
• The failure of many cultures to plate with a high
  efficiency at low cell densities may be due, in
  part, to the dilution of one or more autocrine
  and homocrine factors, and this is part of the
  rationale in using conditioned medium or feeder
  layers to enhance plating efficiency.

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• As the maintenance and proliferation of
  specialized cells, and the induction of their
  differentiation, may depend on paracrine and
  endocrine factors, these must be identified and
  added to differentiation medium.

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• Heterotypic combinations of cells may be,
  initially at least, a simpler way of providing
  the correct factors in the correct matrix
  microenvironment, and analysis of this
  interaction may then be possible with blocking
  antibodies or antisense RNA.

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INITIATION OF THE CULTURE

• Briefly, a culture is derived either by the
  outgrowth of migrating cells from a fragment of
  tissue or by enzymatic or mechanical dispersal of
  the tissue.
• Primary culture is the first in a series of
  selective processes that may in the end give rise
  to a relatively uniform cell line.
• In primary explantation, selection occurs by
  advantage of the cells capacity to migrate from
  the explant, whereas with dispersed cells, only
  those cells that both survive the disaggregation
  technique and adhere to the substrate or survive
  in suspension will form the basis of a primary
  culture.

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• If the primary culture is maintained for more
  than a few hours, a further selection step will
  occur.
• Cells that are capable of proliferation will
  increase, some cell types will survive but not
  increase, and yet others will be unable to
  survive under the particular conditions of the
  culture.
• Hence, the relative proportion of each cell type
  will change and will continue to do so until, in
  the case of monolayer cultures, all the available
  culture substrate is occupied.

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• It should be realized that primary cultures,
  although suitable for some studies such as
  cytogenetic analysis, may be unsuitable for other
  studies because of their instability
• Both cell population changes and adaptive
  modifications within the cells are occurring
  continuously throughout the culture, making it
  difficult to select a period when the culture may
  be regarded as homogeneous or stable.

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• After confluence is reached (i.e., all the
  available growth area is utilized and the cells
  make close contact with one another), cells whose
  growth is sensitive to contact inhibition and
  density limitation of cell proliferation will
  stop dividing, while any transformed cells, which
  are insensitive to density limitation, will tend
  to overgrow.

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• Keeping the cell density low (e.g., by frequent
  subculture) helps to preserve the normal
  phenotype in cultures such as mouse fibroblasts,
  in which spontaneous transformants tend to
  overgrow at high cell densities.
• Some aspects of specialized function are
  expressed more strongly in primary culture,
  particularly when the culture becomes confluent.
• At this stage, the culture will show its closest
  morphological resemblance to the parent tissue
  and retain some diversity in cell type.

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EVOLUTION OF CELL LINES

• After the first subculture, or passage, the
  primary culture becomes known as a cell line and
  may be propagated and subcultured several times.
• With each successive subculture, the component of
  the population with the ability to proliferate
  most rapidly will gradually predominate, and
  nonproliferating or slowly proliferating cells
  will be diluted out.

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• This is most strikingly apparent after the first
  subculture, in which differences in proliferative
  capacity are compounded with varying abilities to
  withstand the trauma of trypsinization and
  transfer
• Although some selection and phenotypic drift will
  continue, by the third passage the culture
  becomes more stable and is typified by a rather
  hardy, rapidly proliferating cell.

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Senescence

• Normal cells can divide a limited number of
  times hence, cell lines derived from normal
  tissue will die out after a fixed number of
  population doublings. This is a genetically
  determined event involving several different
  genes and is known as senescence.
• It is thought to be determined, in part, by the
  inability of terminal sequences of the DNA in the
  telomeres to replicate at each cell division.
• Telomere is a region of repetitive DNA at the end
  of a chromosome, which protects the end of the
  chromosome from deterioration.

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• The result is a progressive shortening of the
  telomeres until, finally, the cell is unable to
  divide further
• Exceptions to this rule are germ cells, stem
  cells, and transformed cells, which often express
  the enzyme telomerase, which is capable of
  replicating the terminal sequences of DNA in the
  telomere and extending the life span of the
  cells, infinitely in the case of germ cells and
  some tumor cells