Cell Culture Techniques Flashcards
What is the history of cell culture techniques?
- They isolated frog hearts in saline solutions then added different reagents to test the change in frog hearts beats.
- German embryologist; stained it.
- Development of cell culture techniques for growing viruses
- Jones Salk and his team grow poliovirus in monkey kidney cells.
- Henrietta Lachs had a carcinoma and Gey removed these cells and found they lived for a long time. Used for the discovery of polio vaccine and no. of chromosomes in humans.
What does cell isolation depend on?
Depends on the cell types/ tissues we want to isolate
Describe the process of isolating cells from the blood
- Density Centrifugation: Takes advantage of the different cell densities and the density gradient. This will decide the cells we isolate e.g. granulocytes and erythrocytes are denser than the density gradient above so can be extracted.
- Immuno-purification: Use an antibody that binds to the cells of interest (coated beads). Magnetic fields used to extract desired beads.
- Fluorescence-activated cell sorter (FACS): Uses antibodies to isolate cells of interest but also based on size rather than just surface marker.
Describe the process of isolating cells from the solid tissues
- Mechanical and enzymatic disruption is used. This can be mechanical such as tearing tissues; staples.
- After disruption, if interested in one cell type; then a technique such as immuno-purification can be used.
- Explant culture - don’t need to disruption as some cells move automatically.
Positives of primary cells (derived directly from tissues)
- unmodified
- good for personalised medicine: a lot of drug therapies respond better to specific pt cells
Negatives of primary cells (derived directly from tissues)
- Aberrant expression of some genes - unfunctional protein expression.
- Variable contamination
- Limited
- Short life-span
- Inter patient variation
- Difficult molecular manipulation
- Phenotypic instability
Why are cell lines preferred to primary cultures?
As you cannot carry out in vitro analysis in the primary cultures that can be reproduced as it will have different characteristics of other cells.
Where are cell lines produced from?
- Isolated from cancerous tissues (e.g. HeLa cells)
- Derived from primary cultures:
- > Spontaneously from prolonged culture, multiple ill-defined, mutations, transformed phenotype so no need for manipulation
- > Through genetic manipulation and transformation of healthy primary cells so manipulated to make them immortal.
How are cell lines generated?
To generate cell lines we target processes that regulate cellular growth and aging
What are the 3 different proteins that are genetically manipulated to produce immortal cells?
- p53 and pRB which are tumor suppressors
- Telomerase: Telomeres which are at the end of chromosomes that are at short tandem repeats.
What is the function of pRB and p53?
Maintain cell cycle checkpoints and maintain genomic stability
Where are telomerase found and what is their function?
Telomerase are found in stem cells, in gametes, and in cancer cells. When the cells divide over time, the telomeres shorten and eventually cell division stops and causes apoptosis
What is the hybrid limit of telomeres?
Telomeres reach a hybrid limit which is very short in length that the chromosome telomeres get damage. This activates apoptosis when they are too short.
Why pRB and p53 work in tandem with telomerase?
Limiting the function of pRB and p53 means that the cell will not apoptosis leading to generating immortal cells. It will also induce telomerase.
How is telomerase induced? Use SV40 and HPV as examples
Taking advantage of viral ‘oncoproteins’ for example in simian virus-40, the viral proteins large T antigen and small t antigen will target p53 and pRb; likewise, human papilloma virus has E6 and E7 that target p53 and pRb.
How do SV40’s T antigens interact with p53 and pRB?
This can cause increased growth without loss of function of these proteins. They do not inhibit; they interact with the DNA binding domains preventing the interaction of p53 and pRB. However, there are still levels within the cell.
How do the E6 and E7 oncoprotein in HPV interact with p53 and pRB?
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. Inhibits p53 directly and pRB.
How is the cell line activated?
- By introducing the telomerase gene into the target primary cell. Some cells need both introductions of the telomerase gene and inactivation of the pRB/p53 for “immortalisation”.
- E6/E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype.
