Cell Culture Techniques Flashcards
How can you isolate cells from a blood sample using Density centrifugation?
Use density gradient medium
Mix the blood sample with the medium and then centrifuge the mixture.
We can then observe the different layers
More dense - sediment through the medium and therefore isolate them from the bottom later
Less dense - mononuclear - remain in the plasma interface
can then isolate the mononuclear cells
Intermediate layer - full of lymphocytes that have the copies of the germ-line
What order the cells form into after a density centrifugation?
- Plasma
- PMBCs
Density Gradient Medium
Granulocytes and erythrocytes
What other techniques can be used?
Immuno-purification:
Antibodies that bind to the cell surface receptor on the cells of interest are used - an antibody that is complimentary to the antigen of interest
The antibodies are coated with magnetic beads
They are then mixed with the sample
The coated antibodies will only bind to the cells of interest
By using a specific magnetic field - can isolate the cells that express the specific antigen
Fluorescence activated cell sorter - FACS
Based on physical properties
can isolate cells by cell surface markers and by size of the molecule but using fluorescence
How can you isolate cells from solid tissues?
Interested in Haematopoietic cells
A the tissue is not liquid you need to use mechanical enzymatic disruption
- this is done by passing the tissue between different needles that then disrupts the cells
- then use enzyme that digest e.g trypsin
What is an Explant tissue?
It is when you use a tissue that has cells that are spontaneously migrating away from the tissue to then isolate the cells of interest
Chondrocytes - migrate away from the cartilage explant
can pick up the chondrocytes and then isolate them
This doesn’t require the disruption techniques
What are primary cells and what are the advantages of using them?
They are cells derived directly from the tissues
- they are unmodified and carry all the genetic information that belong to the specific patient tissue
- they are good for personalised medicine
What are the negatives of using primary cells?
- They express other genes - unfunctional expression
- Variable contamination
- Limited
- Short life span
- Inter-patient variation
- Difficult moleuclar manipulation
- Phenotypic instability - cant carry out in other labs as the characteristics vary between the primary cells therefore not reproducible results
How and why can cancerous tissue be used in primary cultures?
the cells can be isolated from the cancerous tissue - e.g HeLa cells
once derived from primary cell cultures they can be:
- spontaneously grow from the prolonged culture - as they have multiple mutation that cause them to proliferate excessively
- manipulate the cells genetic information - transform the healthy primary cells to make them immortal to use in the lab
What 3 components of cellular growth and ageing are targeted when generating cell lines?
p53
pRb
Telomerase
They are all genetically manipulated to then produce immortal cells
What are p53 and pRb?
They are tumour suppressor genes
They are check points in the cell cycle - they look for genomic stability
p53 checks for:
- unreplicated or damaged DNA after G2
- Chromosome misalignment - after M
- Damaged DNA - after G1
- Unreplicated or damaged DNA - during S
Rb binds to E2F and stops transcription whereas unbound E2F will cause transcription
What is telomerase?
Prevents the fusion of chromosomes and elongates the chromosomes
Every time the cell divides there is shortening of the chromosomes - due to DNA polymerase unable to completely elongate the telomere
As cell division increases the shortening also increases and then there is damage to the chromosomes
This then activated RB causing apoptosis
THEREFORE
The telomerase is used to elongate the telomere in only the cells that need it (where the enzyme is active)
Cells that have activated telomerase:
- Stem cells
- Gametes
- Cancer cells - why they survive and grow infinitely
How do you create immortal cells?
Limit the function of p53 and pRb
Activate telomerase
Some cells need both introduction of the telomerase gene and inactivation of p53 and pRb for immortalisation
How can we inhibit the function of tumour suppressor proteins, or introduce telomerase into cells?
Using viral ‘oncoproteins’
Virus:
- Sv40 = has viral oncoprotein - Large/Small T antigen
- HPV = E6/7
They both target p53 and pRb
They also induce telomerase activity
What do the T-antigens do?
SV40’s T-antigen
Interacts with p53 and pRb protein domain in the DNA - this is the region the p53 and RB usually bind
They stop the p53 and Rb from binding to the binding regions on the domain - there is no tumour suppression
Cause increased growth without the loss of function of the proteins
What do E6/E7 do?
E6 targets p53 for degradation
E7 binds to pRb and inactivates it
Cell lines made by using these oncoprotein are believed to maintain a differentiated phenotype
How is telomerase introduced into the target primary cell?
It is introduced into cells by the telomerase gene
How are plasmids used in cell line production?
The plasmid with the gene for selection and the growth promoting gene is made - it is used to transfer the primary cells with them
- antibiotic resistance is also mixed into the plasmid
The plasmid is then transfixed onto the plate
Selection pressures are then added - can be a antibiotic
The plasmids should be antibiotic resistant therefore if they have transfected properly they will survive in the cell culture as they will be resistant
What are the positive aspects of cell lines?
- Good growth characteristics - standard media
- Phenotypic stability
- Define population
- Molecular manipulation readily achieved
- Good reproducibility
- Good model for basic science
What are the conditions and requirements for growth in culture?
Handled under ascetic conditions - ethanol is used to clean everything
Grown on tissue culture treated plastic flasks/dishes - the plates can be of different size
Maintained at 37 degrees with humidified atmosphere - 8% CO2
- need these conditions to represent the human conditions
An ideal supplemented medium
- needs to be replaced by fresh one every 2/3 days = metabolites and nutrients to reach the cell and to remove debris and waste
- should be the perfect pH, have the growth factors and nutrients
What happens when the requirements and conditions do not occur?
Lack of any of the conditions leads to arresting of the cells - they will stop growing
They will be alive but they won’t proliferate and divide
What is Phenol Red and what is it used for?
It is a medium pH indicator
The balance between metabolites and waste is represented by the colour
The colour of the medium changes based on the presence of metabolites
Yellow - acidic
Red/purple - basic
Red - neutral
What is an adherent cell?
Cells that grow attached to a solid surface
It is anchorage dependent
Agitation is not required
Trypsinisation is required
Tissue culture treated vessels are required
Yield is low
Growth is limited by the surface area
Types of cells - most types of cell lines and primary cultures
What is a suspension cell?
Cells that grow suspended in a liquid medium
They are anchorage-independent
Continuous agitation is required
Trypsinisation is not required
Tissue culture treated vessels are not required
Yield is high
Growth is limited by the concentration of cells in the medium
Types of cells - some non-adhesive cell lines such as hematopoietic
How can cell cultures be contaminated?
- Microbial contamination
- Bacteria
- Yeast
- Fungus
- Mycoplasma
- Virus - Cell lines
- Poor tissue culture technique
- Culture of multiple cell lines at one time
- Accidental mixing of cell lines
What kind of changes do the microbial contaminations cause?
Bacteria
- pH change
- Cloudiness
- Precipitation
- Stink
Yeast
- Cloudiness
- pH change
Fungus
- Spores furry growth
- pH change
Mycoplasma
- they often convert
- Poor cell adherent
- Reduced cell growth
Virus
- Can sometimes be cytopathic
What are the negatives of using cell lines?
- Often lose differentiated function
- Cell substrate interactions dominate
- Does not mimic real tumour conditions
- Lacks cells heterogeneity
- Phenotype needs to be validated
What is an example of a new in vitro model?
3D models - the cell culture is now 3D
Can be a 3D spheroid culture or an 3D organdie culture
What are the differences between organoid and spheroid cultures?
Organoids are derived from stem cells whereas spheroids are derived from cell line monoculture
- organoids are good for drug resistance studies
- spheroids are good to observe the same result
Organoids are from multiple cell lineages whereas spheroids only represent single/partial tissue components
Organoids also reacpitualte organ physiological parameters whereas spheroids only resemble cell organisation
Organoids also are used fro long term culture whereas spheroids are difficult to maintain long term
How do organoids allow the study of cancer drug resistance?
Tumour biopsy is taken
Then turn into an organdie cell culture
- the biopsy can be genome profiled to see if there are any new drug targets
Then treat the organoids with the new drug/treatment
- if there is resistance you can do mutational analysis by undergoing genomic profiling
Then use the treated/passed organoids for survival analysis, histology or optical metabolic imaging
What are the advantages of organoids?
- Gene expression is similar to in vivo methods
- Cell-cell communication re-established
- Cells are orientated in same ways as tissue
- Ideal platform for individualised therapeutic screening
What are the limitations of organoids?
- Limited amount of tissue in some cases
- Organoids in the same culture as heterogenous
- Absence of immune cells in culture system
- Unable to mimic in vivo growth factors/signalling gradients - therefore cant replicate the exact conditions and get the accurate results
What is transfection?
It is the process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods
- includes chemical and physical methods in the lab
Using viral methods = infection
What is lipofection?
Method uses the cationic nature of the head of liposomes
liposomes - small vesicles that are surrounded by bilayer of phospholipids
The lipid heads are positive and the DNA is negative - introduce the DNA into the liposomes and they are then taken up into the cells by endocytosis
- Lipoplexes are positively charged and the membrane is negatively charged - interaction of the lipoplex with the cell membrane of cell
- taken up by endocytosis
- lipoplex is released from the endosome
- transport to the nucleus
- entry to the nucleus is inefficient and may need mitosis
can make it tissue specific by attaching antigens to the liposomes - make sure the liposomes only go to the cells of interest
- can be potential drug carriers for drug delivery
What is electroporation?
The cells with the plasmid DNA are in a medium with plates of a capacitor
High electric field - forms pores which then reseal
- The plasmid DNA then enters the cell when the pores are opened
The rate of pore sealing is dependent on the temperature
What is nucleofection?
Combination of electroporation and lipofection
- lipofection can be toxic to cells sometimes
increased efficiency particularly of non-dividing cells - the transfection is straight into the nucleus - faster
Technology is protected under patent
Different solution and portals are used for each cell type
What is viral infection?
- Exploits the mechanism of viral infection.
- High transfection efficiency.
- Retrovirus, Adenovirus,
but most commonly Lentivirus
are used. - Target cells need to express
the viral receptor to work. - There are safety aspects to
Consider.
