8 Enzymes in cell culture-Peer Abdul Faheem M-6357.pptx

Enzymes used in cell culture

1. Enzymes used in cell culture and the factors affecting growth of cells Submitted by : Dr.Peer Abdul Faheem Roll no. : M-6357 MVSc 1 st year DIV. of veterinary IMMUNOLOGY

2. Cell culture Cell culture can be defined as the process of cultivating cells and tissues outside the body of an organism(invitro) in an artificial environment. Cell culture was first successfully undertaken by Ross H arrison in 1907. Types Primary cell culture Secondary cell culture

3. A primary culture is that type of culture in which inoculating cells are taken directly from the animal tissue via mechanical or enzymatic disintegration methods. There are four stages to consider: Acquisition of the sample, isolation of the tissue, Dissection and/or disaggregation, and Culture after seeding into the culture vessel Secondary cell culture is obtained by subculturing primary cell culture.

4. trypsinization Warm trypsin Whole tissue is being trypsinized at 37◦ C for 4 hours, dissociated cells should be collected every half hour, and the trypsin should be removed by centrifugation and neutralized with serum in medium . Cold trypsin A simple method of minimizing damage to the cells during exposure is to soak the tissue in trypsin at 4◦ C for 6–18 h to allow penetration of the enzyme with little tryptic activity. Following this procedure, the tissue will only require 20–30 min at 37◦ C for disaggregation.

5. Enzymes used in cell culture The most important application of enzymes in the cell culture is the disaggregation of tissues or cells. The enzymes used most frequently for tissue disaggregation are crude preparations of (1) trypsin, (2)collagenase, (3)elastase, (4) pronase , (5) dispase , (6) dnase , and (7)hyaluronidase, alone or in various combinations E.G., Elastase and DNase for type II alveolar cell isolation Collagenase with dispase And collagenase with hyaluronidase

6. Trypsin Obtained from pancreatic cells of swines . Trypsin is inhibited by protease inhibitors such as soyabean. When added to a cell culture, trypsin breaks down the proteins that enable the cells to adhere to the vessel. Trypsinization is often used to pass cells to a new vessel. When the trypsinization process is complete the cells will be in suspension and appear rounded. Collagenase Collagenase cleaves the peptide bonds in native, triple-helical collagen. Because of its unique ability to hydrolyze native collagen, it is widely used in isolation of cells from animal tissue. Elastase It cleaves elastin.

7. Pronase Pronase is a proteolytic enzyme recently introduced as an effective dispersing agent of tissue culture cells. It is found to be a most rapid and complete dispersing agent for primary fibroblastic cell lines and clearly superior to trypsin. Dispase Dispase , a neutral protease isolated from culture filtrates of Bacillus polymyxa , has proven to be a rapid, effective, but gentle agent for separating intact epidermis from the dermis and intact epithelial sheets in culture from the substratum.

8. There are other, nonmammalian enzymes, such as; Trypzean (sigma): TrypZean is a trypsin solution with a completely non-animal origin, intended for cell dissociation. A recombinant maize-derived Trypsin tryple ( invitrogen ). And accutase and accumax also available for primary disaggregation. Accutase ™ is a cell detachment solution comprised of collagenolytic and proteolytic enzymes and does not contain mammalian or bacterial derived products. Accutase ™ is a replacement for trypsin solution and can be used for the passaging of cell lines. Crude preparations are often more successful than purified enzyme preparations, because the former contain other proteases as contaminants, although the latter are generally less toxic and more specific in their action.

9. Trypsin and pronase give the most complete disaggregation, but may damage the cells. Collagenase and dispase , on the other hand, give incomplete disaggregation, but are less harmful. Hyaluronidase can be used in conjunction with collagenase to digest the intracellular matrix, and dnase is used to disperse dna released from lysed cells. Dna tends to impair proteolysis and promote reaggregation

10. Factors affecting growth of the cells Temperature Ph Radiations Osmotic and hydrostatic pressure Ions and salts Oxygen Available water

11. Temperature Temperature is the most important factor that determines the rate of growth, multiplication, survival, and death of all living tissues. High temperatures damage cells by denaturing enzymes, transport carriers, and other proteins. Cell membrane are disrupted by temperature extremes. At very low temperature membranes also solidify and enzymes also do not function properly .

12. Ph   Cell growth is strongly affected by the ph of the medium. Drastic variations in cytoplasmic ph disrupt the plasma membrane or inhibit the activity of enzymes and membrane transport proteins. Maximum growth occurred over a pH range of 7.38 to 7.87. Cell growth declined precipitously on the alkaline side and more gradually on the acid side of the optimal pH range Radiations Ionising rays can produce mutations which may result in death. Radiation works by damaging the genes (DNA) in cells. Genes control how cells grow and divide. When radiation damages the genes of cells, they can't grow and divide any more. Over time, the cells die.

13. Ions and salt: All cells requires metal ions such as K+, ca ++, mg++, fe ++, zn ++, cu++, mn ++ etc to synthesize enzymes and proteins. Most cells do not require Nacl in media however they can tolerate very low concentration of salt. Oxygen Is important gas that affects the growth of cells. Oxygen is required for aerobic respiration. By increasing the fraction of oxygen from 5 to 80% in a graded manner, cell growth is progressively inhibited. Similar graded effects of oxygen on growth of mouse fibroblast and HeLa cells in tissue culture have been reported

14. Available water water is the most essential factor for cell growth. Available water in the culture media determines the rate of metabolic and physiological activities of cells. Sugar, salts and other substances are dissolved in water and are made available for cells.

15. refrences Culture of animal cells by R.Ian Freshney Google Youtube

16. Thank you