Introduction to

Animal Tissue culture

LECTURE OF SUBJECT :

Dr. sharafaldin Al-musawi

College of Biotecholgy

LECTURE: 2

SUBJECT: Animal Tissue culture

LEVEL: 4

Why do we need Cell culture?Research

To overcome problems in studying cellular behavior such as:• confounding effects of the surrounding tissues. • variations that might arise in animals under experimental

stress.Reduce animal use.

Commercial or large-scale production.Production of cell material: vaccine, antibody, hormone.

Initiation of culture

Tissue

Primary culture

Cell line Continuous cell line

Subculture

Stored Stored

Animal Plant

Finite numbers Indefinite numbers

Types of Cell culture

1. Primary Cultures

Derived directly from excised tissue and cultured either as:

Outgrowth of excised tissue in culture

Dissociation into single cells (by enzymatic digestion or mechanical dispersion).

Primary Culture

Preparation

Characteristics of Primary Cultures

5

Primary Culture

Preparation

Characteristics:

Morphologically similar to the parent tissue.

Limited number of cell divisions.

Best experimental models for in vivo

situations.

Advantages & Disadvantages• Advantages:

• usually retain many of the differentiated characteristics of the cell in vivo

• Disadvantages:

• Initially heterogeneous but later become dominated by fibroblasts.

• The preparation of primary cultures is labor intensive.

• Can be maintained in vitro only for a limited period of time.

• Difficult to obtain.

• Relatively short life span in culture.

• Very susceptible to contamination.

• May not fully act like tissue due to complexity of media.

Types of Cell culture2. Continuous Cultures

derived from subculture (or passage, or transfer) of primary culture

Subculture : The process of dispersion and re-culture the cells after they have increased to occupy all of

the available substrate in the culture.

usually comprised of a single cell type.

can be serially propagated in culture for several passages.

There are two types of continuous cultures

Cell lines

Continuous cell lines

Types of continuous culture1) Cell lines

• Cell lines derived from primary cultures have a limited life span.

• After the first subculture, the primary culture becomes cell line.

• finite life, senescence after approximately thirty cycles of division.

• usually diploid and maintain some degree of differentiation.

• It is essential to establish a system of Master and Working banks in order to

maintain such lines for long periods.

Types of continuous culture

2 )Continuous cell lines

can be propagated indefinitely

generally have this ability because they have been transformed by:

tumor cells.

viral oncogenes

chemical treatments

Spontaneously

disadvantage: having very little of the original in vivo characteristics

Transformation VS Transfection

• Transformation

• Spontaneous or induced permanent phenotypic changes resulting from change in DNA

and gene expression that result and effect in:

• growth rate

• mode of growth )loss of contact inhibition)

• specialized product formation

• longevity

• loss of need for adhesion

• Transfection

• Introduction of DNA into a cell (like viral DNA)

Cell Culture Morphology• Morphologically cell cultures take one of two forms:

1. growing in suspension )as single cells or small free-floating clumps) such as: cell lines derived from blood )leukemia, lymphoma).

2. growing as a monolayer that is attached to the tissue culture flask. Such as: Cells from solid tissue )lungs, kidney, breast), endothelial, epithelial, neuronal, fibroblasts.

Hela-Epithelial

MRC5-Fibroblast SHSY5Y-Neuronal

BAE1-Endothelial MCF-7 breast

HT1080- kidney 3LL - lungs

• Excellent model systems for studying:The normal physiology and biochemistry of cells.

The effects of drugs and toxic compounds on the cells.

Mutagenesis and carcinogenesis.

• Used in drug screening and development

• Large scale manufacturing of biological compounds

(vaccines, insulin, interferon, other therapeutic protein)

Cell culture application