Excessive proliferation

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Excessive proliferation Control much studied in
cell culture
Focus cf in vitro tumour
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Trypsin plus EDTA to dissociate cells From each
other, and from ECM
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When dissociated, cells from most tissues round
up. Go spherical, like white blood
cells Observed here by phase contrast microscopy
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Growth medium for mammalian tissue cells
Defined components

• Balanced saline (inorganic ions, including mM
  Ca2)

• Small molecules e.g. essential amino acids,
  vitamins, etc

(Carbon source is glucose glutamine)

• Buffer (CO2 - bicarbonate or HEPES)

• pH indicator (phenol red)

• Antibiotics (cocktail)

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Buffering (pH control) for mammalian cells
Natural buffering, best in culture Is
equilibrium between CO2 and bicarbonate anion
CO2 (g) CO2(s) H2CO3 HCO3-
H
Problem requires special gas phase, air5 CO2
Alternative solutions

• Gas mixture throughout incubator, or sealed flask

• (Some cells only) Replace CO2 with HEPES

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Buffering (pH control) for mammalian cells

• (Some cells only) Replace CO2 with HEPES

HEPES (also PIPES and MOPS) is a Good buffer

• Low toxicity - no inhibition of enzymes
• No interaction with divalent metal cations
• Low temperature co-efficient

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Growth medium for mammalian tissue cells
Defined components

• Buffer (CO2 - bicarbonate or HEPES)

• pH indicator (phenol red)

pH 7.0 healthy
pH 5.0 cells too crowded (or contaminated)
pH 9.0 Sh.. ! We forgot the carbon dioxide!
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Growth medium for mammalian tissue cells
Essential ill-defined component

• 10 (typically) by volume SERUM

Usually foetal bovine serum

Key components of serum

• Adhesion (ECM) proteins Vitronectin and
  Fibronectin

• Growth factors, especially PDGF, also insulin and
  others

PDGF Platelet-derived growth factor
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Growth medium for mammalian tissue cells
What is serum? Difference from plasma?

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Growth medium for mammalian tissue cells
What is serum? Difference from plasma?

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Growth medium for mammalian tissue cells
What is serum? Difference from plasma?

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Growth medium for mammalian tissue cells
What is serum? Difference from plasma?

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Growth medium for mammalian tissue cells
What is serum? Difference from plasma?
Plasma is fluid bathing cells in vivo Added to
culture medium, cells are quiescent

Serum contains contents of platelets
discharged Into the fluid phase as in wound
healing It stimulates replication of many
cell-types in culture
Active ingredients include Platelet-Derived
Growth Factor
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Given suitable fluid medium AND surface, Cells
attach, flatten and acquire characteristic
shapes.
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Pale ring is phase halo - an artefact
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Culture with only chromatin visible
mitoses
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Characteristics of normal tissue cells in
culture which are informative about cancer

• Finite number of division cycles
• Must spread on surface to survive and/or grow
• Require hormone-like growth factors to
  survive/grow

(Note that GF is used in special sense -
signalling)
Each corresponds with an important mechanism
making cells behave well in vivo
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Characteristics of normal tissue cells in
culture which are informative about cancer

• Finite number of division cycles

But what about HeLa cells?
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(HeLa cells are immortal. Cells grown from
tumour)
Cells from normal human tissues divide in culture
for about 9 months, then stop
They cease replication, but do NOT die
Senescence reaching Hayflick
limit Count-down mechanism may be Telomere
shortening
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Basic Cell Culture Terminology
Cells trypsinized from tissue and grown to cover
surface PRIMARY CULTURE
Cells re-trypsinized, diluted and transferred.
Repeated transfers, but will stop SECONDARY
CULTURE
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Basic Cell Culture Terminology
Cells of lab rodents can spontaneously
immortalise (selection in senescent
culture). Human cells have to be genetically
modified, e.g by transfection. (telomerase, SV40
T) Resulting cultures are ESTABLISHED CELL
LINES Establishment in culture models common
event in tumour progression in vivo
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Established lines are useful and can continue to
express differentiated functions
BHK21 Baby Hamster Kidney cells - grow
viruses
MDCK Madin Darby Canine Kidney - study
epithelial polarization
3T3 Mouse cells - study growth control in
culture
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How investigated?
Relation of established lines to actual tumour
cells? Can test by injection into animals - but
is problem of homograft rejection (Use inbred
lines, or thymus-deficient nude mice)

Established lines give tumours but only with
large innoculations, eg. millions of cells
Establishment sometimes called transformation Th
is is confusing !
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How investigated?
Characteristics of normal tissue cells in
culture which are informative about cancer

• Finite number of division cycles
• Must spread on surface to survive and/or grow

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Normal tissue cells in culture do almost
everything (crawling movement, most of cell
cycle) When attached and spread on ECM protein
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Surfaces for growth of animal cells
Surface must become coated with adsorbed
proteins, most important probably vitronectin or
fibronectin
Suitable materials are soft glass and
polystyrene PS must have oxidised surface, to
make it wettable
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Normal tissue cells in culture do almost
everything (crawling movement, most of cell
cycle) when attached and spread on ECM protein
If denied sufficient area of ECM for
spreading Normal epithelial cells apoptose aka
anoikis homelessness Normal fibroblasts
arrest at G1/S restriction point
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This requirement, a feature of normal cells, is
known as Anchorage Dependence Requires positive
signalling via integrins It is lost in many
tumour cells and cultured cells which have been
transformed by oncogene expression (see
Scenario 5) In vivo, loss of this character may
contribute to metastatic capability
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• Loss of anchorage-dependence
• a sensitive test ofmalignant transformation.
• Is closely linked to loss of requirement for
  hormone-like growth factors
• Can be measured experimentally by ability of
  cells
• To form colonies in soft agar.

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How investigated?
Characteristics of normal tissue cells in
culture which are informative about cancer

• Finite number of division cycles
• Must spread on surface to survive and/or grow
• Require hormone-like growth factors to
  survive/grow

Each corresponds with an important mechanism
making cells behave well in vivo Each
property can be lost in culture Paralleling
related changes in actual tumour cells
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Serum contains factors which allow cells to pass
the G1/S restriction point and multiply
3T3 cells need lots. Stop growing when just cover
surface. confluent monolayer
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Because serum Is added, cell culture conditions
resemble wound-healing in vivo
Nuclei of cells in cycle are shown in black
refers to autoradiography with 3HTdR
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Serum requirement can be almost abolished by
infection of cells with RNA tumour viruses by
mutagens by transfection with cDNA isolated from
tumours
In each case this is result of expression of an
activated oncogene. Transformation
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Serum requirement can be almost abolished by
infection of cells with RNA tumour viruses by
mutagens by transfection with cDNA isolated from
tumours
In each case this is result of expression of an
activated oncogene. Transformation
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Thin lawn of normal cells, pale colour Mounds
of transformed cells, dark colour Foci are
clones started by one cell, and so this is like
tumour initiation in tissue
Transformation could be RNA tumour virus, or cDNA
from tumour
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Morphological Transformation
Much higher cell density
Cells often poorly spread
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Morphological Transformation
Transformation is package of changes All result
from loss of requirement for GF signalling Result
of expression of activated oncogene

• Much higher cell density
• Cells often poorly spread
• Colonies in soft agar
• Grow in very low serum

Typically highly tumourigenic e.g. less than
10 cells
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Excessive proliferation Control much studied in
cell culture
Focus cf in vitro tumour