LECTURE OF SUBJECT LECTURE 2 Dr sharafaldin Almusawi

LECTURE OF SUBJECT : LECTURE: 2 Dr. sharafaldin Al-musawi SUBJECT: Animal Tissue culture College of Biotecholgy 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 Plant Animal Tissue Primary culture Subculture Stored Cell line Finite numbers Continuous cell line Indefinite numbers

Types of Cell culture 1. Primary Cultures ØDerived 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 v Characteristics: § Morphologically similar to the parent tissue. § Limited number of cell divisions. § Best experimental models for in vivo situations. 5 Primary Culture Preparation

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 culture 2. 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 v There are two types of continuous cultures � Cell lines � Continuous cell lines

Types of continuous culture 1) 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, senesce 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 � the disadvantage of having retained 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 HT 1080 - kidney MRC 5 -Fibroblast BAE 1 -Endothelial SHSY 5 Y-Neuronal MCF-7 breast 3 LL - lungs

Cell culture application • Excellent model systems for studying: ü The normal physiology and biochemistry of cells ü The effects of drugs and toxic compounds on the cells ü Mutagenesis and carcinogenesis • Used in drug screening and development • Large scale manufacturing of biological compounds (vaccines, insulin, interferon, otherapeutic protein)