Primary cell culture

It's about animal biotechnology Primary cell culture is the ex vivo culture of cells freshly obtained from a multicellular organism, as opposed to the culture of immortalized cell lines. ... When whole or partial tissues are isolated and maintained ex vivo, the procedure is termed primary tissue culture.

1. PRIMARY CELL CULTURE SUBMITTED TO:- MS. SHRUTI GUPTA SUBMITTED BY :- MUSKAAN SHARMA BSC.Hons. BIOTECHNOLOGY 3 rd year

2. WHAT IS PRIMARY CELL CULTURE? A primary cell culture refers to the starting culture of cells, tissues, organs taken directly from an organism. Thus, the primary culture is the initial culture before the first subculture.

3. Criteria for the development of primary cell culture:- Embryonic tissues rather than adult tissues are preferred. Quantity of cells used in the culture should be higher since their survival rate is substantially lower. Tissues should be processed with minimum Damage to cells for use in the culture. The dead cells should ne removed. Selection of appropriate medium. For the addition of serum, FETAL BOVINE source is preferred rather than calf serum or horse serum. Necessary to remove the enzymes used for disaggregation of cells by centrifugation.

4. TECHNIQUES MECHANICAL DISAGGREGATION ENZYMATIC DISAGGREGATION PRIMARY EXPLANT TECHNIQUE

5. MECHANICAL DISAGGREGATION For the disaggregation of soft tissues ( spleen )brain, embryonic liver mechanical technique is employed. Involves careful chopping or slicing of tissue into pieces and collection of spill out cells.

6. CELLS CAN BE COLLECTED BY TWO WAYS FORCING THE TISSUE FRAGMENTS THROUGH A SYRINGE AND NEEDLE. PRESSING THE TISSUE PIECES THROUGH A SERIES OF SIEVES WITH A GRADUAL REDUCTION IN MESH SIZE

7. ADVANTAGES DISADVANTAGES HIGH YEILD RISK OF CELL DAMAGE FASTER THAN ENZYMATIC DISAAREGATION LESS EXPENSIVE QUICK AND SIMPLE. NOTE:- PARTCULARLY USEFUL WHEN THE AVAILABILITY OF THE TISSUE IS IN PLENTY &THE EFFICENCY OF THE YEILD IS NOT VERY CRUCIAL

8. ENZYMATIC DISAGGREGATION Mostly used when high recovery of cells is required from a tissue. It is carried out by using TRYPSIN, COLLAGENASE or some other enzymes .

9. DISAGGREGATION BY TRYPSIN TRYPSINIZATION is commonly used for disaggregation of tissues by the enzyme TRYPSIN. Why crude trypsin is used rather than pure media? Crude trypsin is more effective due to the presence of other proteases. Cells can tolerate crude trypsin better.

10. WARM TRYPSINIZATION Widely used for the disaggregation of cells. Chopped tissue is washed with DISSECTION BASAL SALT SOLUTION (DBSS) then transferred to a flask containing warm trypsin(37c) .

11. COLD TRYPSINIZATION It usually results in a higher yield of viable cells with an improved survival of cells after 24hrs of incubation. This is also called as cold preexposure.

12. DISAGGREGATION BY COLLAGENASE Most abundant structural protein in higher animals. Present in the extracellular matrix of connective tissue & muscle. It is used for human brain, lung & several other epithelial tissues, besides various human tumours &other animal tissues.