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Primary cell culture

The document discusses the preferred use of embryonic tissues for cell culture, emphasizing the need for higher cell quantities and minimal damage during processing. It outlines methods for tissue disaggregation, including mechanical and enzymatic techniques, with a focus on using crude trypsin for optimal cell yield and viability. Additionally, it highlights the importance of selecting appropriate culture media and the role of structural proteins in connective tissues.

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 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.
1. MECHANICALDISAGGREGATION 2. ENZYMATIC DISAGGREGATION 3. PRIMARYEXPLANTTECHNIQUE
 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.
 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
ADVANTAGES DISADVANTAGES HIGH YEILD RISK OF CELL DAMAGE FASTER THAN ENZYMATIC DISAAREGATION LESS EXPENSIVE QUICK AND SIMPLE.
 Mostly used when high recovery of cells is required from a tissue.  It is carried out by using TRYPSIN, COLLAGENASE or some other enzymes.
 TRYPSINIZATION is commonly used for disaggregation of tissues by the enzyme TRYPSIN. 1. 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.
 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) .
 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.
 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.
Primary cell culture