2 - Cell Culture Lecture

7/24/2019 2 - Cell Culture Lecture

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Animal Cell Culture

Diana M. Lopez Ph.D


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I. Characteristics of animal cell culture

II. Growth factors

III. The cell cycle IV. Cell transformation


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Cell culture is the maintenance of cells in vitro .

Cell culture involves taking cells from their natural setting,

characterizing their growth and functional properties, andkeeping them in continuous or semi-continuous culture sothat they are readily available for experimentation.

Growing and maintaining cells is essential for molecularbiology and virology studies. Cultured cells provide the mostpowerful method for cultivation and assay of viruses.

I. CHARACTERISTICS OF ANIMALCELL CULTURE


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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)


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Types of cells

All animal cells are derived from living tissue.Adherent vs. suspension

Cell types1)Primary ce lls

-secondary cells, cell strain, diploid cells

2) Continuous cell lines -transformed cells


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Generation of primary cells

*Primary cells are most conveniently isolatedfrom animal embryos


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Properties of primary cells

Normal chromosome numbers (euploid) and sha pe Require high serum concentration Finite lifespan (30-50 cell divisions) Display properties of differentiated cells Respond to modulators o f cell growth If introduced back into an animal from they which originally

isolated, they m ay s urvive, but will not produce tumors. Adherent cells such as epithelial cells and broblasts need

contact with solid surface for division (anchorage dependentgrowth)

Subject to contact inhibition (will grow again when passaged)


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Anchorage dependent growth


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Fate of primary cells

Senescence (Cells cease proliferation)-Cells undergo crisis after certainnumber of passages

Immortalization (Transformation)


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Properties of continuous cells Fragmented and reduplicated chromosomes(aneuploid) Anchorage independent growth Require only low concentrations o f serum Immortal Do not respond to neighboring cells

Do not display properties of differentiated cells Do not respond to modulators o f cell growth


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Adherent cell lines

Fibroblasts-elongated, spindle s hapeEpithelial cells-polygonal shape


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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 c olony Useful for isolation of mutant cell lines


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Advantages/disadvantages of

continuous cell linesAdvantages Grow rapidly, easy to maintain Can provide large amounts o f virus for

the study of basic aspects of viral replicationDisadvantages Not suitable for studying the subtle effectsof virus infection on cell growth and control

Not appropriate for the study o f differentiatedcell function Not all viruses grow in cell culture


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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


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Change of cell morphology

after viral infection

Herpes Simplex virus(HSV)-induced changes inthe properties of actinmicrolaments o f culturedmonkey broblasts


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Apoptosis versus n ecrosis


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Oncogenic Transformation by Rous

Sarcoma Virus

Transformed Nontransformed


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Cell Junctions

A cell junction (or intercellular bridge) is a type ofstructure that exists within the tissue of some particularmulticellular organisms, such as animals. Cell junctionsconsist of multiprotein complexes that provide contactbetween neighboring cells or between a cell and theextracellular matrix.

Cell junctions are specially important to enable

communication between neighboring cells viaspecialized proteins c alled communicating junctions.


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Types of cell junctions

1) Occluding junctions (tight junctions) -seals adjacent epithelial cells together.2) Anchoring junctions

a) actin lament attachment sitesi. Cell-cell adherens junctions(adhesion belts)ii. Cell-matrix adherens junctions(focal contacts)

b) intermediate lament attachment sites

i. cell-cell (desmosomes)ii. cell-matrix (hemidesmosomes)

3) Communicating junctionsa) gap junctions

b) chemical synapses


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Cell junctions

Focal contact

cadherins

integrins


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Gap junctions

Mediate cell-cell communication

A narrow gap (2-4 nm) between cellscomposed of channel-forming proteinmolecules that allow inorganic ionsand other small water-soluble moleculesto pass directly from the cytoplasm ofone cell to the cytoplasm of another

Gap junctions couple cells electricallyand metabolically


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Gap junctions


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II. Growth factors

Secreted proteins which exert their effects at verylow concentrations (10 -9 to 10 -11 M).

Regulate protein synthesis and cell growth.

Act on cells that express specic receptors.

Can stimulate or inhibit proliferation ordifferentiation.

Over 60 known growth factors-broad specicity (PDGF, EGF)-narrow specicity (erythopoietin)


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Growth factors and their actions


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Growth factorsignaling pathways


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c-myc is a n early response generegulated by growth factors

c-myc g ene was rst discovered in Burkitt's lymphoma p atients

c-myc is a t ranscription factor induced within 15 m in. of growthfactor treatment

c-myc regulates the expression of genes involved in cellproliferation, cell growth, cell differentiation and apoptosis

Malfunctions in c-myc h ave a lso been found in carcinoma of thecervix, colon, breast, lung and stomach


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III. The cell cycle

1) The cell cycle, or cell-divisioncycle, is the series of events thattake place in a cell leading to itsdivision and duplication

(replication).2)Cell cycle can be divided in twoperiods: interphase- duringwhich cells g row, and the m itosisduring which the cell divide intotwo daughter cells.

3) The cell cycle consists of fourdistinct phases: G 1 phase, Sphase, G 2 phase and M phase


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Cell cycle checkpoints

CheckpointsI. G1 checkpoint control

mechanism ensures t hateverything is ready forDNA synthesis.

II. G2 checkpoint controlmechanism ensure thateverything is ready toenter the M phase an ddivide.

III. Metaphase checkpointensures that the cell isready to complete celldivision.

Cell cycle checkpoints are

used by the cell to monitorand regulate the progressof the cell cycle.


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Cells c an enter a n ongrowing G 0 s tate

Serum deprivation of proliferating cellsin culture leads to growth arrest and entryinto a specialized, nongrowing state of G 0

Rate of protein synthesis is dramaticallyreduced in G 0 (20% compared to proliferatingcells)

Cells in G 0 ca nnot progress past the G 1 checkpoint


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Cell cycle synchronization

1)Serum starvation

2) Chemical-induced synchronizationa) nocodazole-arrests cells at metaphase

stage of mitosis.b) hydroxyurea- blocks cells i n S phase.c) mimosine- Late G1 arrest.


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Cyclins/CDKs regulate the cell cycle1. Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases

(CDKs), determine a cell's p rogress t hrough the c ell cycle.2. Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse w on the 2001 Nobel Prize

in Medicine for their discovery of these central molecules

a) Cyclins form the r egulatory s ubunits a nd CDKs t he c atalytic s ubunits o f an activatedheterodimer.

b) When activated by a bo und cyclin, CDKsperform a common biochemical reactioncalled phosphorylation that activates or

inactivates target proteinsc) Different cyclin-CDK combinationsdetermine the downstream proteinstargeted

d)CDKs a re c onstitutively ex pressed in cellswhereas cyclins are synthesised at specic

stages of the cell cycle.

Characteristics


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Regulation of the cell cycle by multiplecyclin/CDK partners


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Regulation of the cell cycle byRetinoblastoma (Rb)

a) Cyclin D binds to CDK4,forming the a ctive C DK4-cyclinD complex.

b) CDK4-cyclin D complexphosphorylate R b.

c) The hyperphosphorylated Rbdissociates from E2F/Rbcomplex, activating E2F.

Rb is a cell cycle regulator protein that binds to and inactivates theE2F transcription factor which regulates the expression of genesrequired for S phase p rogression (cyclin A, E, CDC25 phosphatase)

E2F


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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

IV. CELL TRANSFORMATION


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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 aremeasurable.


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Molecular determinants in the c onversion fromnormal 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


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PROTO-ONCOGENES

Proto-oncogenes are important regulators of biologicprocesses Despite their name, they do not reside in the genome for

the sole purpose of promoting the neoplastic phenotypeThey are essential to normal biologic processes (morethan 100 identified)They play diverse roles in the control of cellulargrowth , including proliferation, apoptosis, genomestability, and differentiation


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ONCOGENES AND TUMORSUPPRESSOR GENES

Proto-oncogenes: control cell division

Tumor suppressor genes: turn off cell division

Mutated alleles, oncogenes, and mutated or losstumor suppressor genes, cause cells to divideuncontrollably


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Oncogenes & tumor suppressors genes

Proliferation genes and antiproliferation genes

An oncogene is a gene that causes the transformation ofnormal cells into cancerous tumor cells, especially a viralgene that transforms a host cell into a tumor cell. In tumorcells, they are often mutated or expressed at high levels.

Proto-oncogene-normal proliferation gene (c-myc)

Tumor suppressor genes-antiproliferation genes found innormal cells. Both copies of a tumor suppressor must bemutated or lost to bring about the loss of growth control.


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Oncogenes & tumor suppressors


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Cellular oncogenes and their functions

Proto-oncogene--->oncogene1) Deletion or point-mutation2) Gene amplication

3) Chromosome rearrangement


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Oncogenes associated with retroviruses

to-oncogene-cogene (retroviralociated)

Mutationsltered expression


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Tumor suppressors

1)Retinoblastoma (Rb)

2) p53 (guardian of the g enome)-regulates the cell cycle- mutated or lost in many cancers-transcription factor (p21 target gene)-can inhibit cell proliferation or induce c ell death-enables c ells to deal with DNA damage


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MECHANISMS OF TUMORSUPPRESSOR GENE INACTIVATION

DeletionPoint mutation

Mutation followed by duplicationLoss of heterozygosityDNA methylationPost-translational mechanism-binding to DNA viral oncoproteins


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Viruses associated with human cancer About 15% of human cancers may arise from a

mechanism involving virusesDNA viruses Associated Tumors Papillomavirus cervical carcinoma

Hepatitis-B virus liver cancer

Herpesvirus family: Epstein-Barr virus Burkitts lymphoma

HHV8 Kaposis sarcomaRNA viruses

Retrovirus f amily:

2 - Cell Culture Lecture

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