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 1ST 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 Harrison in 1907.
Types
1. Primary cell culture
2. 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:
(1) Acquisition of the sample,
(2) isolation of the tissue,
(3) Dissection and/or disaggregation, and
(4) Culture after seeding into the culture vessel
 Secondary cell culture is obtained by subculturing primary cell culture.

5. 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.

6. 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

7. 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.

8. 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.

9. There are other, nonmammalian enzymes, such as;
1. Trypzean (sigma):TrypZean is a trypsin solution with a completely non-animal
origin, intended for cell dissociation.
2. A recombinant maize-derived Trypsin tryple (invitrogen).
3. 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.

10.  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

11. FACTORS AFFECTING
GROWTH OF THE CELLS
 Temperature
 Ph
 Radiations
 Osmotic and hydrostatic pressure
 Ions and salts
 Oxygen
 Available water

12. 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.

13. 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.

14. 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

15. 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.

16. REFRENCES
 Culture of animal cells by R.Ian Freshney
 Google
 Youtube

17. THANK YOU