Cell Culture Techniques Flashcards
As a recap, what are the different ways to isolate cells from tissues?
There are different ways:
You can form a gradient of cells via centrifugation, then place the cells there and centrifuge again. The cells will disperse and form different layers based on the cell density and size, from which you can select the ones you want.
Immunopurification is further purification after density centrifugation. We mix the cells with antibody-coded magnetic beads; the antibody coded is one that is expressed by your selected cell. The magnetic beads bind to the cell that express this marker, and you can then isolate your cells with a magnet.
We can also use a machine called a fluorescence activated cell sorter (FACS). When you have a mixture of cells, you can add to them an antibody with a fluorescent dye on it; this antibody would again be for your desired cell. The bar vibrates, aligning the cells in a single file. They then pass through a laser detector one cell at a time. The ones that fluoresce are the ones that you want, and they can be counted and quantified. They then pass through a series of electromagnets that sort the differently charged cells into a container (fluorescent cells are positively charged).
Briefly, how would you make a cell line from a primary cell culture?
As we learnt before, primary cells have a finite lifespan.
To make them more usable, we have to put them through a method called transfection, and then selection.
We would then characterise them extensively, to make sure they do not have any weird mutations. We do that using methods such as STR profiling, karyotyping, etc.
Then we can use them as culture systems.
How do we upkeep the growth of cells in culture?
- the cells are handled under aseptic conditions
- the cells are grown on treated plastic
- they’re maintained in a warm humidified atmosphere
The growth medium supplies the cells with essential nutrient, antibiotics for treating any infection, growth factors and serum to trigger them to grow, etc.
When cells grow, they secrete by-products which are toxic, so you need to change the growth medium regularly to keep the cells healthy. When the growth medium turns yellow, it means that the pH is not optimal anymore and the medium needs to be changed.
List the advantages and disadvantages of using primary cells.
ADVANTAGES:
- they are unmodified, so simulate in-vivo conditions
DISADVANTAGES:
- they have aberrant expression of some genes, and you can’t tell via microscope
- there is variable contamination
- they have poor growth characteristics (50-100 divisions)
- there is inter-patient variation, so you cannot make a conclusion generalisable unless you use multiple samples
- there is phenotypic instability
- molecular manipulation is difficult
How are cell lines an improvement on primary cell cultures?
- they have good growth characteristics
- they have phenotypic stability
- they have a defined population
- molecular manipulation can be readily achieved
How do we make cell lines?
They can be isolated from cancerous tissues (e.g., HeLa cells)
They can also be derived from primary cultures:
SPONTANEOUSLY: from prolonged culture, multiple ill-defined mutations, transformed phenotype
THROUGH GENETIC MANIPULATION:
through transformation of healthy primary cells
How would we generate cell lines through genetic manipulation?
To generate cell lines, we target processes and proteins that regulate cellular growth and ageing.
Telomerase is present and active only in germ cells, some adult stem cells and most cancers –
they’re not present in somatic cells.
As a recap, describe how telomeres are regulated by p53.
We have telomere-binding proteins that sit at the ends of chromosomes and protect the telomere during replication. Telomere binding proteins recruit and regulate telomerase to ensure an appropriate length of structural DNA is maintained. As we age, the telomeres get shorter and shorter. Soon, they get so short that the telomerase is lost.
When the telomere is exposed, p53 binds to it and triggers growth arrest and cell death.
List some examples of viral oncoproteins.
Examples of viral oncoproteins include (from the):
SIMIAN VIRUS-40
- Large T-Antigen
- Small T-Antigen
HUMAN PAPILLOMA VIRUS (HPV)
- E6
- E7
All of these oncoproteins target p53 and pRb, the senescence regulators in our cells.
Describe how the viral oncoproteins function.
SV40’s T-antigen interacts with p53 and pRb and binds to it. It doesn’t change the functions of the protein itself, it simply doesn’t let it do its job. Thus, this can cause increased growth without loss of function of these proteins.
E6 targets p53 for degradation, and E7 binds to pRb, inactivating it.
Thus, cell lines that are made using E6/ E7 oncoproteins are believed to maintain a differentiated phenotype, so we can use them to study function.
Thus, how would we genetically ‘immortalise’ a cell?
In addition to modifying the cellular senescence regulators of the cell, the telomerase gene (hTERT) can also be introduced into a target primary cell.
Some cells need both introduction of the telomerase gene and inactivation of the pRb for “immortalisation”.
Practically, how would you generate a cell line?
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, e.g. enzyme that can break down toxic compound like antibiotics) and growth promoting gene (e.g. large T-antigen, E6/E7)
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
- Our hope is to incorporate the growth promoting gene into host’s genome, so every time cell divides it will express these genes
COLONIES SELECTED
- Colonies will form from cells that have successfully taken up GPG
- Colonies selected can be grown individually, and can then be investigated
What are some hurdles we have to overcome with generating a cell line?
- Getting DNA into the cells – DNA is toxic, and is negatively charged
- Getting cells to stably incorporate DNA once inside
What are some different methods of transfection?
- CaPO4 co-precipitation
(efficiency improved by Ca2+ nanoparticles) - Lipofection
(cationic lipid transfection systems) - Electroporation
- Viral transfection
- Nucleofection
Describe the method of lipofection.
It’s a method of injecting DNA into cell via a liposome (an artificially prepared vesicle composed of lipid bilayer).
It uses positively charged lipids (cationic head group linked to a hydrophobic tail) and combined with negatively charged genetic material, leads to a net positive charge (so it can ‘mask’ the negative charge of DNA – more effective transfection)
The positive liposome interacts with cell membrane and is taken up by endocytosis (here, it needs to escape the endosomal pathway; if it doesn’t, the method has failed).
The DNA is released from endosome (as the liposome breaks down). The DNA is transported to the nucleus.
Entry to the nucleus is inefficient (small amount taken up, and even smaller amount stably incorporated).
