Cell culture techniques Flashcards
What is cell/tissue culture?
= lab method (in vitro) by which cells are grown under controlled conditions outside their natural environment
The advantages:
- Control the environment - pH, temperature etc.
- Control the micro-environment of the cells - matrix, cell-cell interactions
- Cells can easily be characterised and stained
- Stored in liquid nitrogen for long periods
- Can easily be quantified
- Cheaper to maintain
- Reduced use of animals
What are primary tissue cells? - (type of cells in culture)
- Derived directly from tissues/patients (unmodified)
- Good for personalised medicine
- Finite lifespan - 6/7 divisions, after they die
- Cells divide and/or differentiate - presence of stem cells
- Cells carry out normal functions
What are the methods of isolation for primary tissue cells?
- Cells allowed to migrate out of an explant (natural)
- Mechanical (sieving, pipetting) and/or enzymatic dissociation
**Haemopoietic cells are already individual cells circulating in the blood - you just need to centrifuge them into different densities so heaviest is on the bottom
What are disadvantages of primary tissue cells?
- Inter-patient variation
- Limited number
- Finite lifespan and hard to maintain
- Phenotypic instability
What are immortalised cell lines? - type of cell in culture
- Less limited number of cell divisions or unlimited
- Phenotypically stable, defined population
- Limitless availability
- Easy to grow
- Good model for basic science
- Can be frozen to be moved between labs
What are the methods of production for immortalised cell lines?
1, Isolated from cancerous tissues (can grow quickly)
- Immortalisation of healthy primary cultures (through genetic manipulation
* To generate cell lines we target processes that regulate cellular growth and aging
What happens to telomeres as cells divide over time?
As cells divide over time, telomeres shorten, and eventually cell division stops –> apoptosis
Apoptosis is regulated by p53 and pRb
But if you stop inactivation of p53 and pRb then you wont get apoptosis and the telomere will stabilse:
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?
Taking advantage of viral oncoproteins which target p53 and pRb
- SV40’s T-antigen interacts with p53 and pRb - causes increased growth without loss of function of these proteins
- E6 targets p53 for degradation and E7 binds to pRb inactivating it
- Cell lines made using E6/E7 oncoproteins are believed to maintain a differentiated phenotype
What is immortalisation in cells?
= The ability of a genetically engineered cell line to reproduce indefinitely
- need introduction of telmerase gene and inactivation of the pRb/p53
How can you introduce telomerase to cell lines?
- Making a plasmid with a gene for selection and a growth promoting gene eg. Telomerase
- Introduce the plasmid and transfect the plasmid in the pool of primary cells from a patient tissue
- Treat the cells with the antibiotic (gene from selection from the plasmid), and only those will be able to survive if they have the growth promoting gene
What is the difference bewteen 2D and 3D cultures?
3D cell culture = artificially created environment in which cells are permitted to grow or interact with their surroundings in all 3 dimensions
What is the difference between spheroids and organoids?
Spheroids
- generated from cell lines
- composed of 1 or more cell types that grow and proliferate
- they may exhibit enhanced physiological responses but do not undergo differentiation or self-organisation
- ‘non-stem cells’
Organoids
- generated from primary tissue
- dervied from either PSCs, neonatal tissue stem cells or adult progenitors
- cells spontaneoulsy self-organise into proper differentiated functional cell types
How do organoids allow the study cancer drug resistance?
What is transfection?
= process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods - used in 2D
What is lipofection?
- method of transfection
- introduces DNA to the cell by liposomes
- Easily merges with membrane as they have phospholipid bilayer
- They are net positive, and membrane of cells are negative
What is electroportation?
- method of transfection
- Electric field is applied to cell of interest and will increase its permeability
- Allows drugs/DNA to be introduced into the cell
What is viral infection transduction?
- method of transfection
- Exploits the mechanism of viral infection
- Highly efficient
- Target cells need to express the viral receptor to work
