Animal Cell Culture

Diana M. Lopez Ph.D

I. Characteristics of animal cell culture

II. Growth factors

III. The cell cycle

IV. Cell transformation

I. CHARACTERISTICS OF ANIMAL
CELL CULTURE
Cell culture is the maintenance of cells in vitro.
Cell culture involves taking cells from their natural setting,
characterizing their growth and functional properties, and
keeping them in continuous or semi-continuous culture so
that they are readily available for experimentation.
Growing and maintaining cells is essential for molecular
biology and virology studies. Cultured cells provide the most
powerful method for cultivation and assay of viruses.

Cell culture medium

Culture medium approximating blood plasma

Amino acids
Vitamins
Glucose
Buffers (sodium bicarbonate)
Serum (from calves)
Antibiotics
Other additives (depends on cell type)

Types of cells
All animal cells are derived from living tissue.
Adherent vs. suspension
Cell types
1) Primary cells
-secondary cells, cell strain, diploid cells
2) Continuous cell lines
-transformed cells

Generation of primary cells

*Primary cells are most conveniently isolated

from animal embryos

Properties of primary cells

Normal chromosome numbers (euploid) and shape
Require high serum concentration
Finite lifespan (30-50 cell divisions)
Display properties of differentiated cells
Respond to modulators of cell growth
If introduced back into an animal from they which originally
isolated, they may survive, but will not produce tumors.
Adherent cells such as epithelial cells and fibroblasts need
contact with solid surface for division (anchorage dependent
growth)
Subject to contact inhibition (will grow again when passaged)

Anchorage dependent growth

Fate of primary cells

Senescence (Cells cease proliferation)
-Cells undergo crisis after certain
number of passages
Immortalization (Transformation)

Properties of continuous cells

Fragmented and reduplicated chromosomes
(aneuploid)
Anchorage independent growth
Require only low concentrations of serum
Immortal
Do not respond to neighboring cells
Do not display properties of differentiated cells
Do not respond to modulators of cell growth

Adherent cell lines

Epithelial cells-polygonal shape

Fibroblasts-elongated, spindle shape

Cell clones
Cells in a cell line are often not identical
Cells can be cloned to ensure genetic uniformity
A single cell is isolated and allowed to proliferate
to form a large colony
Useful for isolation of mutant cell lines

Advantages/disadvantagesof
continuouscelllines
Advantages
Grow rapidly, easy to maintain
Can provide large amounts of virus for
the study of basic aspects of viral replication
Disadvantages
Not suitable for studying the subtle effects
of virus infection on cell growth and control
Not appropriate for the study of differentiated
cell function
Not all viruses grow in cell culture

Effect of virus infection on cells

Cytopathology (change in host cell)
Change in morphology
Apoptosis
Necrosis
Cell fusion (syncytia)
Hemagglutination
Change in growth or lifespan
Oncogenic transformation

Change of cell morphology

after viral infection

Herpes Simplex virus

(HSV)-induced changes in
the properties of actin
microfilaments of cultured
monkey fibroblasts

Apoptosis versus necrosis

Oncogenic Transformation by Rous

Sarcoma Virus

Transformed

Nontransformed

Cell Junctions
A cell junction (or intercellular bridge) is a type of
structure that exists within the tissue of some particular
multicellular organisms, such as animals. Cell junctions
consist of multiprotein complexes that provide contact
between neighboring cells or between a cell and the
extracellular matrix.
Cell junctions are specially important to enable
communication
between
neighboring
cells
via
specialized proteins called communicating junctions.

Types of cell junctions

1) Occluding junctions (tight junctions)
-seals adjacent epithelial cells together.
2) Anchoring junctions
a) actin filament attachment sites
i. Cell-cell adherens junctions
(adhesion belts)
ii. Cell-matrix adherens junctions
(focal contacts)
b) intermediate filament attachment sites
i. cell-cell (desmosomes)
ii. cell-matrix (hemidesmosomes)
3) Communicating junctions
a) gap junctions
b) chemical synapses

Cell junctions

cadherins

integrins
Focal contact

Gap junctions
Mediate cell-cell communication
A narrow gap (2-4 nm) between cells
composed of channel-forming protein
molecules that allow inorganic ions
and other small water-soluble molecules
to pass directly from the cytoplasm of
one cell to the cytoplasm of another
Gap junctions couple cells electrically
and metabolically

Gap junctions

II. Growth factors

Secreted proteins which exert their effects at very
low concentrations (10-9 to 10-11M).
Regulate protein synthesis and cell growth.
Act on cells that express specific receptors.
Can stimulate or inhibit proliferation or
differentiation.
Over 60 known growth factors
-broad specificity (PDGF, EGF)
-narrow specificity (erythopoietin)

Growth factors and their actions

Growth factor
signaling pathways

c-myc is an early response gene

regulated by growth factors
c-myc gene was first discovered in Burkitt's lymphoma patients
c-myc is a transcription factor induced within 15 min. of growth
factor treatment
c-myc regulates the expression of genes involved in cell
proliferation, cell growth, cell differentiation and apoptosis
Malfunctions in c-myc have also been found in carcinoma of the
cervix, colon, breast, lung and stomach

III. The cell cycle

1) The cell cycle, or cell-division
cycle, is the series of events that
take place in a cell leading to its
division
and
duplication
(replication).
2) Cell cycle can be divided in two
periods:
interphaseduring
which cells grow, and the mitosis
during which the cell divide into
two daughter cells.
3) The cell cycle consists of four
distinct phases: G1 phase, S
phase, G2 phase and M phase

Cell cycle checkpoints

Cell cycle checkpoints are
used by the cell to monitor
and regulate the progress
of the cell cycle.

Checkpoints
I.

G1 checkpoint control
mechanism ensures that
everything is ready for
DNA synthesis.
II. G2 checkpoint control
mechanism ensure that
everything is ready to
enter the M phase and
divide.
III. Metaphase checkpoint
ensures that the cell is
ready to complete cell
division.

Cells can enter a nongrowing G0 state

Serum deprivation of proliferating cells
in culture leads to growth arrest and entry
into a specialized, nongrowing state of G0
Rate of protein synthesis is dramatically
reduced in G0 (20% compared to proliferating
cells)
Cells in G0 cannot progress past the G1
checkpoint

Cell cycle synchronization

1) Serum starvation
2) Chemical-induced synchronization
a) nocodazole-arrests cells at metaphase
stage of mitosis.
b) hydroxyurea- blocks cells in S phase.
c) mimosine- Late G1 arrest.

Cyclins/CDKs regulate the cell cycle

1. Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases
(CDKs), determine a cell's progress through the cell cycle.
2. Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize
in Medicine for their discovery of these central molecules

Characteristics

a) Cyclins form the regulatory subunits and

CDKs the catalytic subunits of an activated
heterodimer.
b) When activated by a bound cyclin, CDKs
perform a common biochemical reaction
called phosphorylation that activates or
inactivates target proteins
c) Different cyclin-CDK combinations
determine the downstream proteins
targeted
d) CDKs are constitutively expressed in cells
whereas cyclins are synthesised at specific
stages of the cell cycle.

Regulationofthecellcyclebymultiple
cyclin/CDKpartners

Regulation of the cell cycle by

Retinoblastoma (Rb)
Rb is a cell cycle regulator protein that binds to and inactivates the
E2F transcription factor which regulates the expression of genes
required for S phase progression (cyclin A, E, CDC25 phosphatase)

E2F

a) Cyclin D binds to CDK4,

forming the active CDK4-cyclin
D complex.
b) CDK4-cyclin D complex
phosphorylate Rb.
c) The hyperphosphorylated Rb
dissociates from E2F/Rb
complex, activating E2F.

IV. CELL TRANSFORMATION

Generation of transformed cells
(mechanisms of cell transformation)

Culturing of primary cells for long periods

(rodent cells)
Mutagenesis
Tumor viruses (HTLV-I)
Transfection with oncogenes
Can be isolated from tumors

Qualities of transformed cells

Immortalization
Aberrant growth control
-loss of contact inhibition
-anchorage independence (colony formation
in soft agar)
-tumorigenicity (tumor formation in animals)
Malignancy (formation of an invasive tumor in vivo)
* Note: Transformation is a multistep genetic process,
and varying degrees of transformation are
measurable.

Molecular determinants in the conversion from

normal to the malignant cellular phenotype:
Hallmarks of cancer
Growth signals

Self-sufficiency in growth signals

Insensitivity to anti-growth signals

Cell division

Limitless potential for cellular replication

Escape from apoptosis

Oncogenesis

Metastasis and tissue invasion

Sustained angiogenesis

PROTO-ONCOGENES

Proto-oncogenes are important regulators of biologic

processes
Despite their name, they do not reside in the genome for
the sole purpose of promoting the neoplastic
phenotype
They are essential to normal biologic processes (more
than 100 identified)
They play diverse roles in the control of cellular
growth, including proliferation, apoptosis, genome
stability, and differentiation

 ONCOGENES AND TUMOR

SUPPRESSOR GENES
Proto-oncogenes: control cell division
Tumor suppressor genes: turn off cell division
Mutated alleles, oncogenes, and mutated or loss
tumor suppressor genes, cause cells to divide
uncontrollably

Oncogenes & tumor suppressors genes

Proliferation genes and antiproliferation genes
An oncogene is a gene that causes the transformation of
normal cells into cancerous tumor cells, especially a viral
gene that transforms a host cell into a tumor cell. In tumor
cells, they are often mutated or expressed at high levels.
Proto-oncogene-normal proliferation gene (c-myc)
Tumor suppressor genes-antiproliferation genes found in
normal cells. Both copies of a tumor suppressor must be
mutated or lost to bring about the loss of growth control.

Oncogenes & tumor suppressors

Cellular oncogenes and their functions

Proto-oncogene--->oncogene
1) Deletion or point-mutation
2) Gene amplification
3) Chromosome rearrangement

Oncogenesassociatedwithretroviruses

oto-oncogene--->
ncogene (retroviral
sociated)
Mutations
Altered expression

Tumor suppressors
1) Retinoblastoma (Rb)
2) p53 (guardian of the genome)
-regulates the cell cycle

- mutated or lost in many cancers

-transcription factor (p21 target gene)
-can inhibit cell proliferation or induce
cell death
-enables cells to deal with DNA damage

MECHANISMS OF TUMOR
SUPPRESSOR GENE INACTIVATION
Deletion
Point mutation
Mutation followed by duplication
Loss of heterozygosity
DNA methylation
Post-translational mechanismbinding to DNA viral oncoproteins

Viruses associated with human cancer

About 15% of human cancers may arise from a
mechanism involving viruses
DNA viruses

Associated Tumors

Papillomavirus

cervical carcinoma

Hepatitis-B virus

liver cancer

Herpesvirus family:
Epstein-Barr virus
Burkitts lymphoma
HHV8
Kaposis sarcoma

RNA viruses
Retrovirus family:
HTLV-I
adult T-cell leukemia