Cell culture techniques Flashcards
What is density centrifugation of blood?
- Density centrifugation
o Gradient formed by centrifugation
o Cells/sample placed on top of gradient and centrifuged
o Each subcellular component will move up or down when centrifuged until it reaches a position where its density matches its surroundings and then will move no further distinct bands formed most dense at bottom
o If you use blood can isolate different blood cell population using density medium using density centrifugation method. Advantage of diff density of diff cell pop. e.g Ficoll (density of 1.077 g/ml).
o Mix sample with Ficoll after centrifugation we can see the different layers.
- Granulocytes / RBCs are denser than mononuclear cells, the sediment with the density gradient medium so we can isolate them from the bottom layer of the differential layers.
- Less dense mononuclear usually remain in the top layer of the plasma.
- For example lymphocytes can try to isolate the white layer (buffing coat), all lymphocytes usually stay there.
What is FACS?
o Label surface of cells with specific marker for particular cell type
o Laser used to detect which cells are labelled
o Labelled cells diverted to particular tube (see flow cytometry)
o Can isolate cells based on size and markers
What is immuno-purification?
o Antibodies attracted to magnetic beads
o Antibody binds to antigen on cell surface of protein
o Magnet used to attract proteins with magnetic beads purified (other unwanted proteins filtered out)
How do we isolate from solid tissues?
o Mechanical and enzymatic disruption (e.g. collagenase, dispase, trypsin)
o Can also isolate cells using explant culture – tissue harvested in aseptic manner, often minced, and pieces placed in cell culture dish containing growth media. Over time progenitor cells migrate out of the tissue and onto the surface of the dish. (these cells can then be further expanded and transferred into fresh dishes)
o e.g. placenta, the haemopoietic stem cell can diff into any blood cell lineage.
How do we maintain good cell cultures?
o Isolated and maintained under aseptic conditions – reducing possibility of contamination, protecting cells and individual researching (in case cells from humans are infected)
o Cells grown on treated plastic – plates positively charged (as cells negative), also contains ECF proteins, growth media
o Maintained in warm humidified atmosphere
o Antibiotics also added to prevent contamination
o Glutamine added for eliminating ammonia
o Factors important for growth: temperature, pH, nutrients, GFs (via serum or recombinant proteins)
What are the pros and cons of primary cells?
o Derived directly from tissue
o Positive aspects:
+ Good for personalised medicine
+ Unmodified
o Negative aspects:
- Aberrant expression of genes (genetic errors more likely if cultured for longer)
- Variable contamination
- Poor growth characteristics, esp. human primary cells
- Interpatient variation – variation in responses of cells likely
- Phenotypic instability – e.g. loss of receptors after hours of isolation, etc
- Molecular manipulation difficult
What are the pros and cons of cell lines?
o Positive aspects: \+ Good growth characteristics. Standard media \+ Phenotypic stability \+ Defined population \+ Molecular manipulation readily achieved \+ Good reproducibility \+ Good model for basic science o Negative aspects: - Often lose differentiated function - Cell-substrate interactions dominate - Does not mimic real tumour conditions - Lacks cells heterogeneity - Phenotype needs to be validated
How do we produce cell lines?
Production of cell lines:
o Isolated from cancerous tissues (e.g., HeLa cells, can survive by them self without manipulation, however they can be manipulated to become immortal).
o Derive from primary cultures
o Can be spontaneous:
o From tumours or prolonged culture – cells tend to be poorly differentiated, since abnormal, something has triggered them to keep dividing
o Multiple ill-defined mutations – longer we work with culture, more likely to find out mutation
o Transformed phenotype
o From genetic manipulation:
o Transformation of healthy primary cells
o To generate cell lines, we target processes that regulate growth and ageing, examples:
o P53 – antioncogene/TSG
o Retinoblastoma gene (Rb) – antioncogene/TSG
o Telomerase
Telomerase is an enzyme that elongates the chromosomes. In each cell division there is telomere shortening.
Telomeres: short tandem nucleotide repeats at the end of each chromosomes they help maintain stability and stop fusion with other chromosomes.
As cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (p53,pRb)
Hayflick limit- when number of division reaches critical.
What cells have an active telomerase?
Stem cells
Gametes
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?
o Taking advantage of viral ‘oncoproteins’. Target p53 and pRB.
o Interacting with DNA bonding protein domains. By interacting with those domains hey stop p53/pRB from interacting with the domains. Still present in cells and functional.
o SV40’s T-antigen interacts with p53 and pRb. This can cause increased growth without loss of function of these proteins
o E6 targets p53 for degradation, and E7 binds to pRb inactivating it
o Cell lines made using E6/ E7 oncoproteins are believed to maintain a differentiated phenotype.
Why target p53 and Rb?
o They are tumour suppressor genes
o Regulate different parts of cell cycle
o P53 mutated or missing in many cancers
o Many have established that cell lines have lost a functional p53
o Rb inhibits DNA synthesis and arrests cells in G1 of the cycle
How p53 works in a normal cell:
o Telomeres at ends of DNA get shorter every time replication happens
o There are telomere binding proteins at the end to protect telomere
o When telomeres get short, the binding protein is lost (when point of senescence is reached)
o P53 can bind to unprotected chromosomes
o Activated p53 triggers growth arrest or cell death
How can using viral genes stimulate growth?
o Examples: t and T-antigen of Simian Virus-40, E6/7 protein of Human Papilloma virus (HPV)
o T-antigen interacts with normal p53 and Rb – increase growth without loss of function
o HPV – E6 targets p53 for degradation, E7 binds to Rb (HPV more functionally stable)
o Cells made using E6/7 are believed to maintain a differentiated phenotype
How can using telomerase simulate growth?
o Most adult human cells don’t have telomerase that can increase length of telomeres
o Having telomerase will therefore extend lifespan of particular cell – binding protein will remain at ends and no place for p53 to bind
o Therefore: increase expression of telomerase blockage of p53 generate cell line.
o - The telomerase gene can also be introduced into a target primary cell.
o - Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for “immortalisation”
o E6/ E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype
How do we generate a cell line?
o TRANSFECTION (delivery of DNA or RNA into eukaryotic cells):
- Use plasmid with gene for selection (gene that allows us to select for cells that have successfully taken up GPG (growth promoting gene), e.g. enzyme that can break down toxic compound like antibiotics) and growth promoting gene (e.g. large T-antigen, E6/E7)
o SELECTION PRESSURE ADDED
- Toxic compound (e.g. antibiotic) added to culture
- If cell has taken up gene for selection, it will therefore successfully divide as it can break down toxic compound, unsuccessful cells will die
- We then know these cells have also successfully taken up GPG
- Hope is to incorporate growth promoting gene into host’s genome, so every time cell divides it will express these genes
o COLONIES SELECTED
- Colonies will form from cells that have successfully taken up GPG
- Colonies selected can be grown individually, can then be investigated
