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Introduction: the use of animal cell culture

What is cell culture?

Cells removed from animal tissue or whole animals,

will continue to grow if supplied with nutrients and

growth factors

Cells are selected and maintained as independent

manner

Cells in culture may be genetically identical or

genetic variation

Tissue culture means:The ability to survive and grow tissuesoutside the body in an artificial environment.

Embryo Brain Dissociate cells

Brief History:

• In 1902, Leo Loeb placed a

fragments of the skin from the

embryos of guinea pigs in agar

and in coagulated serum and

inserted them into adult guinea

pigs.

• He observed wandering and mitosis of

the epithelial cells.Pathologist Leo Loeb

1869 - 1959

• In 1907, Ross Harrison discovered a way to grow cells outside the body.• Harrison’s first tissue culture:

Biologist, Ross Harrison 1870 - 1959

• At that time, "tissue culture" was a curiosity but in 1998, it was named as one of "medicine’s ten greatest discoveries".

• Alexis Carrel and his colleagues are considered who actually built on Harrison’s idea and laid the main principles for culturing tissues in an artificial media.

• They successfully solved three important problems that faced others before.

• These problems include culture vessels, growing media and death of cultured tissue.

Surgeon, Alexis carrel 1873 - 1944

The history of cell culture

1880

Roux maintain embryonic chick cells in saline solution

1900

Harrison grow for nerve cells by ‘hanging drop’ technique

1940

The antibiotics penicillin and streptomycin are added to

culture medium

Earl’s isolate mouse L fibroblast

Enders grew poliovirus on cultured human cells

1950

Gey culture HeLa Cells

Eagle developed a chemically defined culture medium

1960

Hayflick and Moorhead showed that human cells have a finite life span

Ham grew cells in serum free medium

Harris and Watkins fuse human an mice cells

1970

Kohler an Milstein produce an antibody secreting hybridoma

Sato developed serum-free media from hormones and growth factors

1980

Human insulin was produced from bacteria

Recombinant tissue plasminogen activator was produced from animal

cells

Freshney, Animal cell Culture

Freshney, Animal cell Culture

Why grow animal cells in culture?

To investigate the normal physiology or biochemistry of

cells

To test the effects of compounds on specific cell types

To produce specific artificial tissue by combining

specific cell types in sequence

To synthesize valuable products from large scale cell

culture

Major differences in vitro

1. Specific cell interactions characteristic of the histology of the tissue are lost

2. Heterotypic interactions are lost

3. Loss of homeostatic regulation e.g. endocrine and nervous system

Definition of types of tissue culture

1. Organ culture *kept three dimensional structure of cells *uses fresh sample *cell structure should be supported by gel grid or raft

2. Primary explant culture *cells dissociated by enzyme or mechanical method

3. Cell strains( cell lines) *transformed by primary culture i) monolayer: anchorage dependent ii) suspension: anchorage independent

Freshney, Animal cell Culture

Applications of cell culture in modern biotechnology

1. Production of monoclone antibody

2. Viral vaccine production

3. DNA recombination

4. Pharmaceutical industry

5. Drug activity investigation

6. Tissue transplant( skin or congenital defect…)

7. Clinical investigation (amniocentesis,chromosome analysis)

Studies in cell culture:

1. Intracellular flux e.g. RNA , hormone, metabolites, signal transduction, membrane traffic

2.Intracellular activity e.g. DNA transcription, protein synthesis, energy metabolism, drug metabolism

3.Environmental interaction e.g. infection, drug action, ligand receptor interaction, carcinogenesis.

4. Cell-cell interactione.g. embryo induction, metabolite cooperation, cell proliferation, contact inhibition/density limitation of growth,paracrine growth and differentiation, matrix interaction, invasion

5. Cell products e.g. product formation, exocytosis

6. Genetics e.g. genetic analysis, genetic manipulation/

intervention,transformation, immortalization

Intracellular activity: DNA trancription, protein synthesis, energy metabolism, drug metabolism, cell cycle, differenciation, apoptosis

Cell products:secretion, biotechnology, bioreactor design, product harvesting, down strean processing

Genetics:genetic analysis, transfection, infection, transformation, immortalization,senescence

Cell-cell interaction: morphogenesis, paracrine control, cell proliferation kinetics, metabolic cooperation, cell adhesion and mobility, matrix interaction, invasion

Environmental Interaction:infection, drug action, ligand receptor interactions, cytotoxicity, mutagenesis, carcinigenesis

Intracellular Flux:RNA, hormone receptors, metabolites, calcium, signal transduction, membrane trafficking

More recent development

The three major categories of valuable products from animal cells:

Viral vaccines

Monoclonal antibody

Recombinant glycoproteins

Stem cell studies

Tissue engineering

Advantages of Tissue Culture

1. Control of environment, consistency and reproducibility e.g. pH, temp, pressure, O2, CO2

2. Characterization and homogeneity of sample

3. Economy, scale and mechanization, less expensive reduction of animal use

4. In vivo modeling delivering of specific compound, regulation of concentration, duration of exposure time

5. Easier to deal with virus contamination compared to the animal experiment

Disadvantages of cell culture

1. Expertise

2. Quantity

3. Differentiation and selection characteristic change, adaptation to nutrient change

4. Origin of cells

5. Instability

Biology of Cultured Cells

I. The culture environment

the influence of environment on culture cells is

expressed in four ways:

1. The nature of substance or phase in which the

cell grow

e.g. plastics, semisolid( gel, or agar), or liquid

2.The physiochemical and physiological institution

of medium

3. The constitution of gas phase

4. The incubation temperature

Major differences of cultured environment and animal model:

In Vivo In Vitro

1. some cell type proliferate cells does proliferate 2. cell/cell interacts loss of cell interaction 3. hormones and nutrient no effects of hormone affect

inoculation

Spreading 24hrs

1-2 hrs

Serum derived glycoprotein Cell surface glycoprotein Conditioning factor

1.Cell adhesion

Cell adhesion Proteins Three classes of transmembrane protein

I. . Mediate interactions between homologous cells Cell-cell adhesion molecule

CAMs( calcium dependent)-calmodulin cadherins

II. Mediate cell-substrate interaction Integrin matrix protein receptor

III. Interact with matrix Proteoglycan

CAM

Basement membrane

Connective tissue/stroma

cadhedrinsCell layer

integrin proteoglycan

S

G2

M

G1Gap2

Mitosis

DNA synthesis

Gap1

Nuclear oncogene: Myc

Cyclins CDK kinase

Cyclins CDK kinase

Receptor kinase: EGFR, erbB

2. Cell Proliferation

cell cycle is divided by four stages

S

G2

M

G1Gap2

Mitosis

DNA synthesis

Gap1

Rb/E2F

P53 mutation

Check point

Restriction point

Cell Cycle Arrest

DNA repair or Apoptosis?

P53 mutation

S

G2

M

G1

Gap2

Mitosis

DNA synthesis

Gap1

Restriction point

Check point

Rb-P

p53+

p53+

Cell Cycle Progression

Control of proliferation of cell culture by:

a. signals from the environment

e.g. growth factors, EGF, FGF, PDGF………

b. Intracellular control

cyclins, Rb gene products

c. cell membrane receptors

link intra and extra cellular pathway

3. DifferentiationDedifferentiation:: caused the inability of cell lines to express in vivo phenotype

dedifferentiation may occur due to

1) undifferentiated cell of the same lineage over grow terminally differentiated cell or reduce proliferative capacity

2) the absence of the appropriate inducers ( hormones; cell or matrix interaction) cause deadaptation

Differences between dedifferentiation, deadaptation and selection

1) Dedifferentiation

specialized properties of cells are lost irreversibly

e.g. Hepatocyte: loss of enzyme activity of

arginase, aminotransferase, could not store

glycogen or secret serum proteins

2) Deadaptation

products re-induced by certain culture environment

hormones, cell/cell interaction, cell/matrix

interactions….)

e.g. induction of tyrosine aminotransferase in

hepatocyte by floating collagen raft)

3) Selection

isolation of cultured cell type by specific methods

e.g. confluent feeder layer or selection media for epidermal cells

e.g. use of D-valine containing medium for growth of epithelium

Stem cell

Progenitor cell Differentiated cell

conmmitment

Stem cell regeneration

t=24-36hrs t=18-72hrs

attenuation amplification

Differentiation

Stem cell

Progenitor cell

Stem cell regeneration

t=18-72

amplification

t=18-72

Differentiation

Regulatory adaptation

Differentiation

amplificationattenuation

Initiation of culture

Cells form primary culture if:

1. Survive the disaggregation process

2. Adhere to the substrate or survive in suspension

Cell lines may be established if cells are capable of proliferation

Freshney, Animal cell Culture

Elements of selection in the evolution of cell lines

factors influencing selectionstage primary explant enzymatic disaggregationisolation mechanical damage enzymatic damage

primary culture adhesion of explant; cell adhesion and outgrowthspreading migration

first subculture trypsin sensitivity;nutrient,hormone,and substrate limitation

propagation as a cell relative growth rates of different cells;selective line overgrowth of one lineage;nutrient’hormone,and substrate limitations;effect of cell density on predominance of normal and transformed phenotype

senescence normal cells die out;transformed cells overgrowtransformation

Characteristics of continuous cell lines

1. Chromosomes are usually aneuploid

2. Chromosome numbers are always between diploid and tetraploid

3. Cells forming continuous cell lines are tansformed or preexisted

4. A number of properties of continuous cell lines are associated with malignant transformation

A cell culture contain:

multiple stem cell

undifferentiated but committed cell

mature differentiated

CHO cell: Chinese hamster Ovary

PC12 cell

HeLa cell

Breast Cancer

MCF

Melanoma cell

B16 F10