Objective

To establish and maintains of primary cell culture of chick embryo and zebra fish embryo.

Theory

Tissue culturing as a technique was first used almost 100 years ago to elucidate some of the most basic question in the developmental biology. Cell culture is an exceedingly precious tool for investigators in numerous fields. The animal cell was first introduced by Ross Harrison in 1907. It facilitates analysis of biological processes and properties which are not readily accessible at the level of the intact organism. To getting the cells for culture must be isolated from a donor animal tissue or organ. The cell culture may be divided into three according to their history: 1) primary, 2) secondary and 3) continuous cell culture. The primary and secondary cells are usually diploid cells.  Primary cell lines derived directly from an intact tissue like animal’s embryo or kidney. Secondary cells are derived from primary cultures. And the continuous cell lines are usually derived from malignant tissue.

Cells are taken directly from the living tissue and established for the growth invitro in primary cell culture. These cells are undergone very few population doublings and are more representative of the major functional component of the tissue from which they are derived in comparison to continuous cell lines  thus representing a more representative model to the in vivo studies. Primary cells from different species may be used allowing highlighting potential differences between humans and preclinical test species. Before in vivo studies rats or mouse cells can be used to refine doses and reduce the number of animals required for preclinical toxicology. Human cells can be used to determine accuracy of extrapolating human data from an animal model.

Primary cultures consist of cells before sub culturing is carried out. In primary cultures either grown as explants cultures or subjected to enzyme treatment and the dissociated cells grown as monolayer. This represents a mixture of cell types present in the tissue; they are closer to the tissue composition present in vivo. Primary cultures are used to study, maintenance of functional ability of cells and effect of external stimuli of cell functions, cell-cell interactions. When sub culture, cells with better growth potential, those losing the property of cell- cell interactions necessary to maintain the functional state of the tissue.

Fertile chick embryo (9-11 days) and zebrafish (12-24 hours) are used as a convient cell system to grow a number of human pathogenic viruses. The usefulness derives from many types of different tissues found in the eggs, tissues that will support the growth of different viruses. The use of chicken egg is expensive and the use of cell culture is preferred possible. The use of chick embryo for the production of vaccine is necessarily subject to many controls. The eggs are free from specific pathogens and originate from healthy flocks. The processing of the fertilized eggs must be conducted under aseptic conditions in an area where no other infectious agents or cells are handled at the same time.

The Advantage of using zebrafish is easy to maintain and breed and the generation time is relatively short.  To study vertebrate development are the mutations are relatively easy to obtain and to screen. The zebrafish embryo is transparent and small size.
 

Principle

For this experiment the fertile Chick and Zebrafish embryo has the greatest advantage because these embryo is accessible at all stages following lying of the egg. Early blastoderms can be cultured in vitro for enough to form a recognizable primary body plan. Furthermore it is possible to explants pieces of tissue onto the chorioallantoic membrane (CAM) of advanced embryo, where they become vascularzed and will grow and differentiate in effective isolation.

From tissue fragments cells are dispersed by proteolytic enzyme like trypsin and mechanical shake. After washing the cells, they are suspended in the growth medium and distributed in petri plate, test tubes, and bottles. The cells adhere to glass surface and grow out to form monolayer sheet. The cell culture relies the growth of cells in a semi confluent monolayer attached to the surface. To subculture, the cells are separated from the monolayer or relevant tissue usually with trypsin to form suspension of the single cells. These suspended cells are then used to seed a new plate. Following growth at 37ºC, cells will multiply, attached to the surface and form a new monolayer within a few days. The media used to grow the cells consists of basic nutrients and salts supplemented with serum to provide the growth factor, but the composition is varies depend up on the cell types. Antibiotics and antifungal are included to prevent the bacterial growth and fungal contamination in such a rich growth medium. For Zebrafish embryo culturing L-15 medium is widely used. L-15 (Leibovitz) medium was basically formulated for use in CO2 free system requiring sodium bicarbonate supplement.L-15 is buffered by its complement of salts, free base amino acids and glactose substituted for glucose to help maintain physiological pH control.

Application

1. Cultured cells are used for reconstruction of damaged tissue or replacement of non- functional cells or tissues.
2. Easy growing of cells invitro has led to a spurt in the activity to apply cell culture technology to synthesize or produce a variety of bimolecules at an industrial scale.
3. To study the processes taking place in animal cells, e.g., metabolic regulations, cell physiology.
4. Cultivation of patient’s own cells in vitro in order to generate enough cells to genetically manipulate the cells to replace a missing function, to overcome problem of immune rejection and of scares tissue source.
5. Use of tissue culture avoids many ethical objections raised against animal experiments and also allows experiments on human tissues which otherwise could not be done in vivo.

Disadvantages

1. Tissue culture requires strict aseptic conditions and skilled person and techniques.
2. Cost of animal tissue culture and labor, often may be high and prohibitive.
3. The growth of metabolic regulatory mechanism that exist under in vivo conditions are absent in culture condition
4. Cell lines may not reflect the actual condition in vivo and results may not be reproducible in the living animals.
5. Tissue culture may not always be possible.