Cellular pathology: Cell Culture Techniques Flashcards
1
Q
Explain the different ways that cells can be isolated from blood?
A
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Density centrifugation
- Uses a density gradient medium and a centrifuge to seperate components of blood based on density
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Immuno-purification
- Involves coating magnetic beads with a specific antibody that will bind to an antigen present on surface of cells of interest within a mix of cells.
- Once bound you can use magnetic fields to attract the cell of interest as it will be magnetic due to binding of antibody coated magnetic beads
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Fluorescence activated cell sorter (FACS)
- You add a fluorescent marker to a specific antibody.
- These fluorescent antibodies are added to a mixture of cells containing cell of interest.
- Fluorescent antibody will bind to an antigen on surface of cell of interest thus making it fluorescent.
- Mix of cells put through cell sorter and pass through one at a time.
- Cells pass through laser detector allowing fluorescent cells and to be counted and quantified.
- They then pass through a series of electromagnets that sort the differently charged cells into a container (fluorescent cells are positively charged).
2
Q
Explain the different ways that cells can be isolate from solid tissue
A
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Mechanincal and enzymatic disruption
- Mechanical disruption involves using scalples or passing tissue through series of needles
- Enzymatic involves use of enzymes, e.g. dispase, trypsin or collagenase, to break down tissue
- Immuno-purification can then be used to isolate specific cells of interest once mechanical/enzymatic disruption has occured
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Explant culture
- E.g. chondrocytes can be isolated from a cartilage explant culture as they migrate from the cartilage as they both grow
3
Q
What are some advantages of using primary cells compared with cell lines?
A
- Unmodified
- Good for personalised medicine - can be used to check if cells of a patient respond well to a drug based on their genetic profile
4
Q
What are some disadvantages of using primary cells compared to cell lines?
A
- Aberrant expression of some genes - Results in production of unfunctional proteins
- Variable contamination
- Short life-span - This means they can’t be cultured for a long time
- Inter patient variation
- Difficult molecular manipulation
- Phenotypic instability
5
Q
Where do the cells that are used to prduce a cell line come from?
A
- Isolated from cancerous tissues (e.g., HeLa cells)
6
Q
In what manner can the cells of cell lines grow?
A
- They can grow spontaneously from prolonged culture via multiple ill-defined mutations that cause those cells to become immortal
- They can also grow through genetic manipulation which transforms healthy primary cells which also causes them to become immortal
7
Q
What are the different pathways that are targeted in order to make the cells of a cell line immortal?
A
- p53
- pRB (retinoblastoma protein)
- Telomerase
8
Q
What is the function of telomerase?
A
- Somatic cells have a finite lifespan (no. of times they can divide) and once they reach that limit they enter cell senescence, cell growth arrest.
- Each time a cell divides the length of the telomeres, the ends of the chromosome that protect it from damage, shorten
- Once length of telomeres reach a critical point chromosome gets damged and p53 and retinoblastoma become activated leading to apoptosis
- Telomerase is an enzyme that extends the length of the telomeres after each round of cell division by adding a telomere repeat sequence to the ends of the telomeres.
- This only occurs in particular cells that express the 2 subuits of telomerase (TERT and TERC).
9
Q
What cells is telomerase active in?
A
- Stem cells (e.g. germline cells)
- Cancer cells - allows them to survive and replicate indefinitely
10
Q
In what way are p53, pRB and telomerase maniuplated in cells in order to make them immortal?
A
- p53 is inhibited
- pRB is inhibited
- Telomerase activity is increased by expressing its subunits: TERT (Telomerase reverse transcriptase) and TERC (Telomerase RNA component)
11
Q
How are p53 and pRB inhibited in cells?
A
- You use viral oncoproteins that target p53 and pRB
- These viral oncoproteins include:
- Large T antigen and small T antigen from the simian virus-40 (SV40)
- E6 and E7 from the Human Papilloma Virus (HPV)
12
Q
How do the large and small T antigens of SV40 inhibit p53 and pRB?
A
- SV40’s T-antigen interacts with p53 and pRb.
- Specifically, they interact with the DNA binding domains of pRB and p53 which prevents p53 and pRB from binding to their targets
- This can cause increased cell growth without loss of function of these proteins
13
Q
How do the E6 and E7 viral oncoproteins of HPV inhibit p53 and pRB?
A
- E6 targets p53 for degradation, and E7 binds to pRb inactivating it
14
Q
How is telomerase activity increased in cells?
A
- The telomerase gene can also be introduced into a target primary cell.
15
Q
Explain how the telomerase gene is introduced into primary cells
A
- Create a plasmid containing a gene for selection (antibiotic resistance marker) AND the gene wanting to be introduced into the cell (telomerase)
- Introduce the plasmid into the primary cells (Transfection)
- The primary cells are grown on a plate with growth medium and then a selection pressure added (antibiotic)
- This will result in ONLY those cells with antibiotic resistance to survive
- These cells produce colonies as they grow which are selected for in order to produce more colonies
16
Q
Do cells only need the increase in telomerase activity or the inhibition of p53 and pRB to become immortal?
A
- Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for immortalisation
Other cells do only need one of the 2 methods to become immortal
23
Q
The pH of growth medium changes due to build up of metabolites, how can you monitor the pH of the growth medium?
A
- Most growth mediums contain a pH indicator, e.g. phenol red, that changes colour depending on the pH of the medium
- When medium is alkaline (pH 7.4-7.6) phenol red is red/purple
- When medium is acidic (pH 6.8) phenol red is yellow
- When medium is neutral (pH 7.0), which is ideal for growth, phenol red is tomato red
26
Q
What are the differences between adherent cells and suspension cells?
A
- Adherent cells are anchorage-dependent while suspension cells are anchorage-independent
- Adherent cells don’t require agitation while suspension cells do
- Adherent cells require trypsinization while suspension cells don’t
- Tissue culture treated vessels are required for adherent cells while they aren’t required for suspension cells
- Yield for adherent cell culture is low while it is high for suspension cell culture
- With adherent cell culture growth is limited by surface area while with suspension cell culture it’s limited by concentration of cells in medium
- Types of cell grown in adherent cell cultures include Most types of cell lines and primary cultures while types of cells grown in suspension cell cultures include Some non-adhesive cell lines such as hematopoietic
31
Q
What are some of the differences between organoid and spheroid 3D in vitro models?
A
- Oganoids are derived from stem cells while spheroids derived from cell line monoculture
- Organoids represent multiple cell lineages while spheroids represent singl/partial tissue components
- Organoids replicate organ physiolocial parameters while spheroids transiently resemble cell organisation
- Organoids can be used for long term culture while spheroids are difficult to maintain long term
32
Q
Why are organoids particularly useful for drug-resistance studies?
A
- Because cells extracted from a patient are still grown in a 3D conformation with the same cell-cell contacts they would have in vivo.
- This allows for more accurate results to be collected on the effects drugs would have on those cells in vivo
35
Q
What is transfection?
A
- Transfection is 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 e.g. a plasmid, a CRISPR/Cas9 complex
37
Q
What are some methods of transfection?
A
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Chemical
- Lipofection
- Calcium phosphate
- Cationic ploymer
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Physical
- Electroporation
- Nucleofection
- Microinjection
38
Q
Explain the process of lipofection
A
- Plasmid DNA is introduced into liposomes, this is becuase negatively charged DNA attarcted to positively charged phosphate head of liposome
- Liposomes then fuse with cell via endocytosis
- The endosomes formed as a result of this endocytosis are then broken down inside the cell
- This releases the plasmid DNA inside the liposome which will then travel to the nucleus of the cell
39
Q
Lipofection can be used for drug delivery, how can this process be made tissue specific?
A
- Can be made tissue specific by attaching tissue specifc antigens to surface of the liposome
40
Q
Explain the process of electroporation
A
- Cell is placed in a solution with the plasmid DNA and the plates of the capacitor, which are charged.
- The high electrical field created temporarily forms pores on the cell membrane of the cell, increasing its permeability.
- This allows the plasmid DNA to go through into the cell
- Once inside plasmid DNA needs to travel to the nucleus
- The rate of pore resealing is dependent on temperature – maintaining a lower temperature after electroporation reduces the rate of pore resealing, allowing more of the plasmid DNA to enter.
