Cellular pathology: Cell Culture Techniques Flashcards
Explain the different ways that cells can be isolated from blood?
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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).
Explain the different ways that cells can be isolate from solid tissue
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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
What are some advantages of using primary cells compared with cell lines?
- 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
What are some disadvantages of using primary cells compared to cell lines?
- 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
Where do the cells that are used to prduce a cell line come from?
- Isolated from cancerous tissues (e.g., HeLa cells)
In what manner can the cells of cell lines grow?
- 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
What are the different pathways that are targeted in order to make the cells of a cell line immortal?
- p53
- pRB (retinoblastoma protein)
- Telomerase
What is the function of telomerase?
- 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).
What cells is telomerase active in?
- Stem cells (e.g. germline cells)
- Cancer cells - allows them to survive and replicate indefinitely
In what way are p53, pRB and telomerase maniuplated in cells in order to make them immortal?
- p53 is inhibited
- pRB is inhibited
- Telomerase activity is increased by expressing its subunits: TERT (Telomerase reverse transcriptase) and TERC (Telomerase RNA component)
How are p53 and pRB inhibited in cells?
- 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)
How do the large and small T antigens of SV40 inhibit p53 and pRB?
- 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
How do the E6 and E7 viral oncoproteins of HPV inhibit p53 and pRB?
- E6 targets p53 for degradation, and E7 binds to pRb inactivating it
How is telomerase activity increased in cells?
- The telomerase gene can also be introduced into a target primary cell.
Explain how the telomerase gene is introduced into primary cells
- 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
Do cells only need the increase in telomerase activity or the inhibition of p53 and pRB to become immortal?
- 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
What are some of the advantages of cell lines compared to primary cells?
- Good growth characteristics. Standard media
- Phenotypic stability
- Defined population
- Molecular manipulation readily achieved
- Good reproducibility
- Good model for basic science
What are some of the disdvantages of cell lines compared to primary cells?
- Often lose differentiated function
- Cell-substrate interaction dominate
- Does not mimic real tumour conditions
- Lacks cells heterogeneity
- Phenotype needs to be validated
What conditions are required for growth in culture?
- Aseptic conditions
- Gloves
- Lab coats
- Work under a hood
- Ethanol spray on all equipment used
- Cells grown on tissue culture treated plastic flasks/dishes
- Cells maintained in a warm (37°C) humidified atmosphere (5% CO2) - optimal growth conditions
- Cells grown in ideal supplemented medium that needs to be replaced by fresh one every 2/3 days
Why is the growth medium important for the growth of cells?
- Provides cells with a no. of essential things they need to grow:
- Correct pH
- Space
- Growth factors
- Antibiotics (Penicillin, Strep)
- Nutrients (L- glutamine)
- Correct Temperature
Give examples of some widely used growth mediums
- RPMI 1640
- DMEM
Why does the growth medium need to be replaced every 2-3 days?
- Because the cells produce metabolites which need to be removed and the depleted nutrients need to be replaced
The pH of growth medium changes due to build up of metabolites, how can you monitor the pH of the growth medium?
- 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
What are adherent cells?
- Cells that grow attached to a solid surface
What are suspension cells?
- Cells that grow suspended (floating) in a liquid medium
What are the differences between adherent cells and suspension cells?
- 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
What are the 2 ways that cell cultures can be contaminated?
- Microbial contamination
- Cell line cross-contamination
What are the different types of microbial contamination? For each type state some signs of contamination
- Bacteria (pH change, cloudiness/turbidity, precipitation, stink
- Yeast (cloudiness, pH change)
- Fungus (spores furry growths, pH change)
- Mycoplasma (often covert, poor cell adherent, reduced cell growth)
- Virus (sometimes cytopathic)
What are some reasons for cell line cross-contamination?
- Poor tissue culture technique
- Culture of multiple cell lines at one time
- Accidental mixing of cell lines
3D in vitro models overcome all of the negatives of cell lines. What are the 2 main types of 3D in vitro model?
- Organoid
- Spheroid
What are some of the differences between organoid and spheroid 3D in vitro models?
- 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
Why are organoids particularly useful for drug-resistance studies?
- 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
What are some advantages of using organoids for patient drug testing?
- Gene expression as in vivo (87% phenotype and genotype similarity)
- Cells-cell communication re-established
- Cells are orientated in same ways as tissue
- Ideal platform for individualized therapeutic screening
What are some disdvantages of using organoids for patient drug testing?
- Limited amount of tissue in some cases (e.g. prostate)
- Organoids in the same culture are heterogeneous
- Absence of immune cells in culture system
- Unable to mimic in vivo growth factor/signalling gradients
What is transfection?
- 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
What is the term used to describe transfection carried out using viral methods?
- Infection
What are some methods of transfection?
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Chemical
- Lipofection
- Calcium phosphate
- Cationic ploymer
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Physical
- Electroporation
- Nucleofection
- Microinjection
Explain the process of lipofection
- 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
Lipofection can be used for drug delivery, how can this process be made tissue specific?
- Can be made tissue specific by attaching tissue specifc antigens to surface of the liposome
Explain the process of electroporation
- 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.
Explain the process of nucleofection
- 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 solution and protocols are used for each cell type
What is viral infection?
- A process that exploits the mechanism of viral infection to introduce foreign DNA into a eukaryotic cell
- Has a high transfection efficiency.
- Retrovirus, Adenovirus but most commonly Lentivirus are used
