Cell Culture Techniques Flashcards
Cell culture
group of cells grown in a nutrient solution from a single original cell
How do we isolate cells?
Isolation of cells depends on the tissue from which we want to isolate them
How do we isolate cell populations in blood?
Density Centrifugation
-density gradient medium was used which separates different cell populations depending on their different densities
Layers of the blood after centrifugation
- Granulocytes & erythrocytes are denser than mononuclear cells, and therefore sediment through the density gradient medium
- The less-dense mononuclear cells usually remain in the top layer (plasma interface). We can isolate the plasma interface and isolate the less dense mononuclear cells
Techniques to isolate specific cells from blood
Immuno-purification
Fluorescence Activated Cell Sorter (FACS)
Immuno-purification
Magnetic beads are coated with an antibody which binds to one cell surface receptor/antigen present in our cells of interest/cells we want to isolate when we mix with blood sample
By application of a magnetic field, we are able to instruct those beads that are attached to the cells of interest
Fluorescence-activated cell sorter (FACS)
this uses antibodies to isolate the cells of interest, but it is also based on other properties of the cell (e.g. physical property), therefore can also isolate cells based on their size (as well as their cell surface markers)
Isolating cells from solid tissues
Mechanical and enzymatic disruption to disrupt the cells from the solid tissue.
Then, magnetic immuno-purification technique used to extract/isolate cells of interest
When do we not need to use mechanical and enzymatic disruption to isolate cells from solid tissues?
In a cartilage explant, the chondrocytes migrate away from a cartilage explant spontaneously. The cartilage explant should just be set on a plate and chondrocytes will isolate by themselves (no need for mechanical enzymatic disruption).
Cell lines
immortalised cells that continue to grow and divide indefinitely in vitro for as long as the correct culture conditions are maintained
Why are cell lines produced?
Due to the disadvantages of primary cells (derived directly from tissues), we are interested in producing cell lines.
Where are cell lines isolated from?
healthy or cancerous tissues
-(e.g. HeLa cells): HeLa cell line include cells extracted from cervical carcinoma
-primary cultures
Cell lines derived from primary cultures can either…
- survive spontaneously by themselves without manipulation
- be genetically manipulated in order to transform them and make them immortal
How are cell lines made immortal?
By the genetic manipulation of 3 different proteins which regulate cellular growth and ageing:
- p53
- pRB
- telomerase enzyme
What are p53 and pRB encoded by?
tumour suppressor genes
p53 and pRB both maintain genomic stability by mediating cell cycle checkpoints
Viruses which target tumour suppressor proteins (p53 & pRB)
Viruses such as SV40 and HPV contain viral oncoproteins which can target tumour suppressor proteins
Mechanism of action of SV40 to inhibit tumour suppressor proteins
SV40’s T-antigen (viral oncoproteins) interacts with the DNA binding domains to which p53 & pRB usually bind. By interacting with these domains, they prevent these proteins from interacting with their domains, and therefore their activity is not carried out. This can cause increased growth. However, there are still levels of p53 and pRB in the cell, meaning they are still functional, but they just can’t bind to their DNA binding domains.
Mechanism of action of HPV to inhibit tumour suppressor proteins
E6 targets p53 for degradation, and E7 binds to pRB inactivating it
How is telomerase transfected into primary cells to immortalise them?
1) Design a plasmid containing a gene for selection (selection marker e.g. antibiotic resistance marker).
2) Insert into the plasmid the sequence of the gene we want to transfect into the primary cell.
3) Once the plasmid construction is completed, the primary cells are transfected with those vectors.
4) We can identify the cells that have been positively transfected by antibiotic selection because we inserted an antibiotic resistance marker. Only the cells that have been positively transfected will express this antibiotic resistance marker, and these cells will be able to survive in an environment containing antibiotic (E.g. neomycin).
How do we know when to change/replace the growth medium?
Most of the universal media contain phenol red, which is a medium pH indicator. Its colour changes based on the pH of the medium. The pH of the medium changes due to the presence of metabolites. We want a neutral pH, which is a tomato red colour.
- it becomes yellow when the medium is acidic
- it becomes purple when it is basic
Adherent cells
cells that grow attached to a solid surface
- low yield
- growth limited by surface area
- most types of cell lines and primary cultures
How to detect microbial contamination?
Under the microscope, black particles moving within the space between cells should attract your attention. Some bacteria show directed movement and actively rush away from their current position.
