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