Cell culture techniques: cell culture Flashcards
What are characteristics of cell lines culture?
Immortalised cells Less limited number of cell divisions (~30) or unlimited They can be grown in 2D or 3D Phenotypically stable, defined population Limitless availability Easy to grow Good reproducibility Good model for basic science
What are the two methods of production for a cell culture ?
- Isolated from cancerous tissues (e.g. HeLa cells)
- Immortalisation of primary cultures:
a) Spontaneously from prolonged culture: multiple ill- defined mutations transformed phenotype
b) Through genetic manipulation: artificial transformation of healthy primary cells
How do you produce cell lines culture through genetic manipulation?
To generate cell lines we target processes that regulate cellular growth and ageing
- p53
- pRb
- Telomerase
What are p53 and pRB encoded by?
tumour suppressor genes
p53 and pRB both maintain genomic stability by mediating cell cycle checkpoints
Function of telomerase
maintains stability and length of telomeres at end of chromosomes to prevent their fusion with other chromosomes, preventing cell senescence
-ONLY in cells with active telomerase (e.g. stem cells, cancer cells)
How can we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions?
- inhibit function of p53 & pRB
- introduce/over-express telomerase
How can we inhibit the function of p53 & pRB?
by taking advantage of viral oncoproteins
Viruses which target tumour suppressor proteins (p53 & pRB)
Viruses such as SV40 and HPV contain viral oncoproteins which can target tumour suppressor proteins
Mechanism of action of SV40 to inhibit tumour suppressor proteins
SV40’s T-antigen (viral oncoproteins) interacts with the DNA binding domains to which p53 & pRB usually bind. By interacting with these domains, they prevent these proteins from interacting with their domains, and therefore their activity is not carried out. This can cause increased growth. However, there are still levels of p53 and pRB in the cell, meaning they are still functional, but they just can’t bind to their DNA binding domains.
Mechanism of action of HPV to inhibit tumour suppressor proteins
E6 targets p53 for degradation, and E7 binds to pRB inactivating it
How do we reach complete immortalisation with the help
telomerase?
telomerase gene -introduced into a target primary cell.
Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for “immortalisation”
How is telomerase transfected into primary cells to immortalise them?
1) Design a plasmid containing a gene for selection (selection marker e.g. antibiotic resistance marker).
2) Insert into the plasmid the sequence of the gene we want to transfect into the primary cell.
3) Once the plasmid construction is completed, the primary cells are transfected with those vectors.
4) We can identify the cells that have been positively transfected by antibiotic selection because we inserted an antibiotic resistance marker. Only the cells that have been positively transfected will express this antibiotic resistance marker, and these cells will be able to survive in an environment containing antibiotic (E.g. neomycin).
What condition are needed for cell growth in a culture ?
Handled under aseptic conditions
- Grown on tissue culture treated plastic flasks/dishes
- Maintained in a warm (37°C) humidified atmosphere (5% CO2)
What are the condition and requirements for an ideal supplemented medium?
What happens if these condition are not reached?
Conditions which are needed for growth: temperature, pH, space, growth factors, serum, antibiotics and nutrients
If these conditions are not met the cell cycle would stop.
medium needs to be replaced every two to three day.
because of:
depletion of the nutrients in the medium
release of waste products into the medium after cell metabolism
How would you detect if the medium needed to be replaced?
Red to purple = basic (pH 7.4-7.6) Fresh medium Tomato red = neutral (pH 7.0) Day 6 Yellow = acidic medium (pH 6.8)
Most of the universal media contain phenol red, which is a medium pH indicator. Its colour changes based on the pH of the medium. The pH of the medium changes due to the presence of metabolites. We want a neutral pH, which is a tomato red colour.
