cell culture techniques Flashcards
Describe the process of density centrifugation and what it is used for
used to isolate cells from blood
uses centrifugation to separate cell types based on their densities
most dense = bottom of tube
least dense = top of tube
In the process of density centrifugation what layers are usually formed after centrifugation of the blood
LEAST DENSE - plasma - PMBCs - density gradient medium - granulocyte, erythrocyte MOST DENSE
DENSITY CENTRIFUGATION
what is usually found in the PMBC layer?
PMBC = peripheral blood mononuclear cells
lymphocytes are usually localised within the PMBC layer
what is immune-purification used for?
how is it different to density centrifugation
used to isolate cells from blood
this method is used to isolate SPECIFIC cell types (in contrast to just density centrifugation)
Immunopurification is BOTH techniques used, in contrast to density centrifugation alone.
- density centrifugation
THEN
- immune purification = FURTHER purification
describe the process of immuno-purification
density centrifugation and THEN immune purification
- Mix cells with antibody-coded magnetic beads. These antibodies coded are specific to the cell type you are trying to extract!
- The magnetic beads bind to the cell that express the specific marker for the antibody
- You can then use a magnetic to isolate your specific cells
describe what FACS is and what it is used for and its process
FACS- fluorescence activated cell sorter
used to isolate cells from blood
- mixture of cells + specific antibody (for the cell you want to isolate) with a fluorescent dye on it
- cells align in a single file and pass through a laser detector, ONE CELL AT A TIME
- the cells that we specifically want will fluoresce and now be positively charged
- they will then pass through electromagnets that sort the cells based on their charge
(only the specific cell you are trying to isolate will be charged!) - cells will be in different containers based on their charges: positive sample (ones we want) and negative sample (ones we don’t want)
name the 3 methods you can use to isolate cells from blood
- density centrifugation
- immuno-purification
- FACS
what does immuno-purification method and FACS have in common
both use antibodies!
what method can be used to isolate cells from solid tissues
You would remove the tissue mechanically (scalpel) and enzymatically (dispase, trypsin, collagenase).
You could then use a method called magnetic immuno-purification of the specific cell you’re looking for to view it.
what is the method used to make a cell line from a primary cell culture
(4)
primary cells have a finite lifespan so to make them more useable we put them through:
- transfection
- selection
- ensure no weird mutations present using STR profiling or karyotyping
- can now be used as culture system to make a cell line
What are some advantages of using primary cells derived directly from tissues in cell line production?
- they are unmodified
- they are good for personalised medicine
(can test is a patients cells respond well to a specific treatment or not, before actually giving them the treatment)
What are some disadvantages of using primary cells derived directly from tissues in cell line production?
- abnormal expression of some genes = abnormal protein production
- variable contamination
- short life span
- inter patient variability (cells derived are not all identical)
- difficult molecular manipulation
- phenotypic instability
where do cell lines come from
These cell lines can be isolated from healthy or cancerous tissues (eg- HeLa cells)
how do these cell lines grow
2
cell lines derived from primary cultures can grow via 2 ways
- grow spontaneously from prolonged culture and become immortal due to the genetic alteration they carry
OR
- grow through genetic manipulation
- transformation of healthy primary cells into immortal cells so that these cells can be used in the labs etc
what are the different pathways that are targeted to make the cell line immortal
(3)
target processes that regulate cellular growth and ageing = generate immortal cell lines
- P53
- PRB
- Telomerase enzyme
p53
encoded by?
function?
tumour suppressor genes
involved in cell cycle checkpoints
maintains genomic stability
pRB
encoded by
function
tumour suppressor genes
involved in cell cycle checkpoints
maintains genomic stability
describe what telomeres are and what happens to them overtime
Telomeres (STR) protect the ends of chromosomes from degradation by nuclear enzymes, therefore maintaining chromosome stability. With each round of replication, the length of the telomere shortens (chromosomal lengthening = telomere shortening).
When the chromosomes reach a threshold length, cells enter cell senescence aka permanent cell cycle arrest.
THIS IS THE NORMAL PROCESS
what can happen to telomeres which then go on to trigger apoptosis and by which mechanism does this apoptosis occur
If cells bypass the. threshold length (because of mutation) the telomeres become critically short resulting in apoptosis via P53 and PRB activation.
what is the role of telomerase enzyme
Telomerase is an enzyme which acts to increase the length of the telomeres (addition of STRs)
In which cells is telomerase found/active
This enzyme telomerase is active in ONLY SPECIFIC CELLS such as stem cells which are self renewing and can continuously divide.
Many cancer cells have active telomerase activity which is why they can continue to survive indefinitely and replicate.
what changes are made to the following pathways to create immortal cells:
- pRB
- pP3
- telomerase activity
INHIBIT:
- P53
- PRB
ENHANCE:
- telomerase activity
All the above changes result in the finite production of primary cells to form a cell line
how can we inhibit prb and p53 in order to produce a cell line
using viral oncoprotein
what are some viral oncoprotein that can be used to target P53 and PRB?
state the virus and the viral oncoprotein
VIRUS = simian virus-40 (SV40) ONCOPROTEIN = large T antigen & small T antigen
VIRUS = HPV ONCOPROTEIN = E6 & E7
describe the mechanism by which SV40 acts to inhibit p53 and pRB
The large T antigen interacts with the proteins (p53 and pRB) DNA binding domain (where p53 and pRB would usually bind).
By the large T antigen binding to the DNA binding domains, it prevents the p53 and pRB from interacting with the DNA binding domain, which means although the proteins are still functional, they cannot carry out their activity.
describe the mechanism by which HPV acts to inhibit P53 and PRB
HPV E6:
- targets p53 for degradation
HPV E7:
- binds to pRB = inactivating pRB
how do we increase telomerase activity
Telomerase (hTERT) can be introduced into a target primary cell
Some cells need both introduction of the telomerase gene and inactivation of the pRB + p53 for immortalisation