Cell Culture Techniques Flashcards
Describe use of skin cell spray
Harvest stem cells for a severe burn
Stem cells sprayed onto burn site
Using cells spray gun device
Cell application heals burn
Describe Immuno-purification
Antibody-coated magnetic beads
Mixed with heterogeneous cells
Isolation of antigen-expressing cells
Describe use of fluorescence activated cell sorters (facs)
Collection process = sample is injected into a stream of sheath fluid that passes through the flow cell and laser intercepts
Stream carries cell through a vibrating nozzle
Disturbance in stream causes it to break into a droplet containing ideally one cell
An electrical charging ring is placed just at the point where the stream breaks into droplets
A charge is placed on the ring based immediately prior to fluorescence intensity being measured, and the opposite charge is trapped on the droplet as it breaks from the stream
The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge
Describe the return of the stream to neutral when using a FACS
In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off
Describe cell isolation from solid tissues
Mechanical and enzymatic disruption using: dispase trypsin collagenase
Then magnetic Immuno-purification
Discuss positives/negatives of primary cells (derived directly from tissues)
+ve:
Unmodified
Good for personalised medicine
-ve: Aberrant expression of some genes Variable contamination Limited Short life-span Inter patient variation Difficult molecular manipulation Phenotypic instability
Describe the methods of cells derived from primary cultures
Spontaneously, from prolonged culture, multiple ill-defined mutationstransformed phenotype
Through genetic manipulation
Transformation of healthy primary cells
What do we target to generate cell lines
- To generate cell lines we target processes that regulate cellular growth and ageing
Effect on telomeres as cells divide
- As cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (p53,pRb)
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?
Answer: taking advantage of viral ‘oncoproteins’
Virus = Simian Virus-40 (SV40) Viral oncoprotein = Large T antigen Small t antigen Cellular targets = p53 + pRb
Virus = Human Papilloma Virus (HPV)
Viral oncoprotein = E6 + E7
Cellular targets = p53 + pRb
Compare the use of SV40’s T-antigen and E6/E7
V40’s T-antigen interacts with p53 and pRb. This can cause 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
Describe what is required for immortalisation of telomeres
- The telomerase gene can also be introduced into a target primary cell.
- Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for “immortalisation”
Effect of E6/E7 + telomerase transformations
🡪 E6/ E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype
List positives of cell lines
Good growth characteristics. Standard media Phenotypic stability Defined population Molecular manipulation readily achieved Good reproducibility Good model for basic science
Conditions and requirements for growth in culture
Handled under aseptic conditions
Grown on tissue culture treated plastic flasks/dishes
Maintained in a warm (37°C) humidified atmosphere (5% CO2)
In ideal supplemented medium that needs to be replaced by fresh one every 2/3 days* (*Phenol red is a medium pH indicator)
