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
Telomerase (hTERT) can be introduced into a target primary cell to INCREASE telomerase activity. But how do we do this?
- Create a plasmid containing a gene for selection (antibiotic resistance marker) AND the gene wanting to be introduced into the cell (telomerase)
- Transfection of primary cells with vector
- Selection pressure added (bacteria). This will result in ONLY those cells with antibiotic resistance to survive and produce colonies
Advantages of cell lines?
- Good growth characteristics
- Phenotypic stability
- Defined population
- Molecular manipulation readily achieved
- Good reproducibility
- Good model for basic science (in vitro)
Disadvantages of cell lines?
- Often lose differentiated function
- Cell-substrate interaction dominate
- Does not mimic real tumour conditions
- Lacks cells heterogeneity
- Phenotype needs to be validated
What are the conditions required for growth in culture?
Handled under aseptic conditions
Grown on tissue culture treated plastic flasks/dishes
Maintained in a warm (37 degrees Celsius) humidified atmosphere (5% CO2)
Grown in an ideal medium which needs to be replaced by a fresh medium every 2/3 days
what are the 2 ideal growth mediums for growth in culture and why do they need to be replaced often
- RPMI 1640
- DMEM
These mediums need to be replaced and a fresh one provided because the cells produce metabolites which need to be removed and because the depleted nutrients need to be replaced
growth in culture conditions
what is meant by handled under aseptic conditions?
Lab coats
Gloves
Ethanol spray on equipment you’re going to use
Work under/inside the hood
growth in culture conditions
what needs to be taken into consideration when forming the ideal medium
- CO2
- pH
- Space
- Growth factors
- Serum (FBS)
- Antibiotic (Penicillin, Strep)
- Nutrients (L- glutamine)
- Temperature
How can you find out the pH of the growth medium and find out if it is the ideal pH?
USING PHENOL RED (medium pH indicator)
IDEAL = TOMATO RED (pH 7)
phenol red is a medium pH indicator. What is meant if the medium is:
- purple
- yellow
- tomato red
why does the pH of the medium change?
purple = basic (ph 7.4-7.6)
yellow = acidic (ph 6.8)
tomato red = neutral (ph 7)
The pH of the medium changes due to the presence of the metabolites
ADHERENT VS SUSPENSION CELLS
definition?
adherent cells:
cells that grow attached to a solid surface
suspension cells:
cells that grow suspended (floating in a liquid medium)
ADHERENT VS SUSPENSION CELLS
anchorage dependent?
adherent cells:
anchorage dependent
suspension cells:
anchorage independent
ADHERENT VS SUSPENSION CELLS
agitation needed?
adherent cells:
not required
suspension cells:
constant agitation required
ADHERENT VS SUSPENSION CELLS
trypsinization needed?
adherent cells:
required
suspension cells:
not required
ADHERENT VS SUSPENSION CELLS
tissue culture treated vessels?
adherent cells:
required
suspension cells:
not required
ADHERENT VS SUSPENSION CELLS
yield
adherent cells:
low
suspension cells:
high
ADHERENT VS SUSPENSION CELLS
growth limited by..?
adherent cells:
by the surface area
suspension cells:
by concentration of cells in the medium
ADHERENT VS SUSPENSION CELLS
types of cells?
adherent cells:
most types of cell lines and primary cultures
suspension cells:
some non-adhesive cell lines such as haematopoietic
what are the 2 types of cell culture contamination
- microbial contamination
2. cell line cross contamination
what are the 5 types of microbial contaminations
- bacterial
- yeast
- fungus
- mycoplasma
- virus
MICROBIAL CONTAMINATIONS
characteristics of bacterial
pH change
cloudiness
precipitation
stink
MICROBIAL CONTAMINATIONS
characteristics of yeast
pH change
cloudiness
MICROBIAL CONTAMINATIONS
characteristics of fungus
pH change
spores furry growth
MICROBIAL CONTAMINATIONS
characteristics of mycoplasma
often not well detected
poor cell adherent
reduced cell growth
MICROBIAL CONTAMINATIONS
characteristics of virus
sometimes cytopathic
what can result in cell lines cross contamination
3
- poor tissue culture technique
- culture of multiple cell lines at one time
- accidental mixing of cell lines
Cell lines cell culture have some disadvantages. However new 3D in vitro models overcome all of these negatives. What are the 2 main types of 3D models?
organoids
spheroids
3D in vitro models
describe organoids
- Derived from stem cells
- Multiple lineages
- Recapitulate organ physiological parameters
- Long term culture
Patient derived organoids allow the study of cancer drug resistance
3D in vitro models
- Derived from cell line monoculture
- Represent single/partial tissue components
- Transiently resemble cell organisation
- Difficult to maintain long term
advantages of organoids
- gene expression as in vivo
- cell to cell communication re-established
- cells are orientated in same ways as tissue
- ideal platform for personalised therapy
disadvantages of organoids
- limited amount of tissue in some cases
- organoids in the same culture of heterogeneous
- absence of immune cells in culture system
- unable to mimic in vivo growth factor/signallings
What is transfection?
The process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the lab
Eg: a plasmid, CRISPR/Cas9 complex
What are the different methods of transfection?
- Lipofection
- Electroporation
- Nucleofection
- Viral infection/transduction
name a method of chemical transfection
lipofection
name 2 methods of physical transfection
- Electroporation
* Nucleofection
name a method of viral transfection
infection
what is lipofecction?
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
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).
what can lipofection be used for
Liposomes can be used for the transfection of drugs (can carry drugs into other cells). These can be tissue specific by attaching different antigens to their surface (of the liposomes). This makes sure that the liposomes only go to the specific tissues of interest and release the drug into that tissue ONLY.
What is electroporation?
The cell is placed in a solution with the plasmid DNA and the plates (which are charged).
forms pores on the cell membrane of the cell, increasing its permeability.
This allows the DNA to go through.
The rate of pore resealing is dependent on temperature – maintaining a lower temperature after electroporation reduces the rate of pore resealing, allowing the plasmid DNA to enter.
What is nucleofection?
It’s a combination of electroporation and lipofection
We punch holes in the cell via electroporation, then DNA enters via the liposome (lipofection) and then enters nucleus.
Different solutions and protocols are used for each cell type.
It has an increased efficiency (but less than viral transfection; however, it’s less hazardous)
What is viral infection/transduction?
The most commonly used are lentiviruses, as they can enter cells not undergoing cell division. You can also use retrovirus and adenovirus.
It exploits the normal mechanisms of viral infection.
It usually has high transfection efficiency
The disadvantage is that viruses used can be harmful to humans (biological scientist).
What can viral infection/transduction be used for
Your put the gene that you want to carry into the cells into the lentivirus (for example). It needs to be the right size so that the virus will be able to package it. You then transfect packaging cells with it. These packaging cells make the virus, so you collect the virus particles from them. You then use those particles to infect your target cells for the transduction and transfection of your gene of interest.
