Cell Culture Techniques Flashcards
1
Q
How do we isolate cells from blood?
A
- density centrifugation
- fluorescence activated cell sorter (FACS) - flow cytometry
- immuno-purification
2
Q
What conditions do you need for growth in culture?
A
- isolated and maintain under aseptic conditions
- Cells are grown on treated plastic
- maintained at optimum temp
- given nutrients and growth factors via the medium
3
Q
What are Primary cells? Give positive and negative aspects?
A
- derived directly from tissue
- Positive aspects - unmodified
- Negatives - abberant expression of some genes (may not mimic normal tissue), ethical issues, poor growth characteristics, variable contamination, inter-patient variation, may have to get tissue from diseased body
4
Q
What is the ideal model?
A
- good growth characteristics
- phenotyping stability
- defined population
- molecular manipulation readily achieved
5
Q
How do we obtain cell lines from tissue?
A
- spontaneous - from tumours or prolonged culture, multiple ill-defined mutations, transformed phenotype (faster cell divides, the less well it works)
- genetic manipulation
6
Q
How do we genetically manipulate a cell to produce a cell line
A
- need to manipulate processes that regulate growth and ageing
- target different proteins in cells that are responsible for regulating growth
- p53, Rb, Telomerase
7
Q
What are p53 and Rb?
A
Tumour suppressor genes
- normally prevent a cell from dividing inappropriately
- regulate different parts of the cell cycle
- p53 binds to the ends of chromosomes when telomeres get too short - triggers cell death/arrest
- p53 is mutated/missing in many cancers
- Rb inhibits DNA synthesis and arrests cells in G1
8
Q
Which Viral genes are commonly used to generate cell lines?
A
- Simian virus-40 - t and T-antigen (interact with normal p53 and Rb - increased growth without loss of function)
- HPV - E6/E7 (E6 targets p53 for degradation, E7 binds to Rb)
9
Q
Transfecting in telomerase
A
- prevents erosion of telomeres
- some cells need it to silence Rb for immortalisation
- E6/E7 and telomerase are believed to maintain a differentiated phenotype
10
Q
Cell line generation process
A
- Introduce foreign DNA
- Plasmid introduced into cell will express growth promoting gene (e.g. T, E6/E7) and gene interested in
- Gene for selection expresses enzyme that will break down an antibiotic. Some cells expressing that gene will not die
- Neomycin will kill all the cells that are not taken up and aren’t expressed
- this will give little colonies which can be separated and grown out
11
Q
Hurdles to overcome
A
- getting DNA into the cells
- getting the cells to stably incorportate the DNA when inside
12
Q
Methods of transfection
A
- Calcium phosphate co-precipitation (not very efficient)
- Lipofection - using cationic lipid transfection systems
- Electroporation - make little holes in membrane through which DNA can diffuse through
- Viral transfection
- Nucleofection (mixture of lipofection and electroporation)
13
Q
Lipofection
A
- Surround DNA with a lipid coat - changes charge of DNA from -ve to +ve and means it can readily fuse with cell membrane
- taken up by endocytosis
- small amount of DNA will migrate to nucleus where it may be incorporated
14
Q
Electroporation
A
High electric field forms little pores which DNA can diffuse through - reseal afterwards
15
Q
Viral transfection
A
- Have a cell that generates viral particles and expresses selection gene and gene of interest - packaged into viral particles (will not replicate until into second cell)
- Placed into target cells - these are the cells you are transfecting, they express a receptor to which the virus can bind
- virus puts its DNA into cell -> integrates with cell’s DNA via reverse transciption
- very efficient
- significant safety implications as you are using viruses
16
Q
Nucleofection
A
- High energy field blows holes in the cell membrane
- then placed in lipid coat which can get into the cells and be transported to the nucleus
- patented technology
17
Q
Long term storage
A
- Cryo-preservation
- problems - ice crystals can form and disrupt cell membrane- need to freeze slowly and thaw fast - use preservatives to remove some of the water (DMSO)
18
Q
Give a disadvantage of cell lines and how you can fix it?
A
- Rapidly dividing cells often lose their differentiated function
- therefore want to try and manipulate cells in a way to retain funciton but to continue growth
- can use heat in some cases to stop the dividing and regain some functions
- changing culture conditions such as ECM, co-culture or 3d culture may also help
19
Q
Example of co-culture
A
- grow VSMCs and Endothelial cells together
- when they group together, they arrange as if they were in vivo (EC outside and VSMC inside)
- cell-cell communication is re-established
20
Q
What are the 3 retroviruses we can use in Viral transfection?
A
- adenovirus
- Associate adenovirus
- Lentivirus
