Cell Culture Techniques Flashcards
1.What is density centrifugation?
-You isolate different cell populations depending on their density in a density gradient medium.
- In a density centrifugation, where would granulocytes/erythrocytes isolate?
They are dense so isolate at the bottom through the density gradient medium
3.Where would lymphocytes isolate in a density centrifugation?
In the white buffer coat
4.How does the process of immuno-purification work?
Magnetic beads are coated with antibodies
Antibodies bind to specific cell surface antigens
Antigens (of interest) are extracted by applying magnetic field
5.How does Fluorescence activated cell sorter (FACS) isolate cells of interest?
Using antibodies
Or
Physical Properties eg size
6.List three mechanisms to isolate cells from blood?
- Density Centrifugation
- Immuno-purifiation
- Fluorescence activated cell sorter
7.How would isolate cells from solid tissues?
First the tissue is either disrupted :
mechanically(scalpels/needles) or
by enzymatic digestion.
The cells of interest can then be isolated by magnetic immuno-purification
8.Give an example of a cell where isolation would be easy?
chondrocytes migrating away from a cartilage explant mean they will separate by themselves
9.What is meant by “primary cells”
Cells derived directly from tissue
10.What is a cell line
Cell line is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space.
11.What are two advantages of using primary cells for treatment?
They are unmodified
-Good for personalised medicine
12.What are 7 disadvantages of using primary cells for treatment?
- Aberrant(abnormal) expression of some genes
- Variable contamination
- Limited
- Short life-span
- Inter patient variation
- Difficult molecular manipulation
- Phenotypic instability
13.How can you make cells immortal?
- Cells are isolated from cancerous tissues (HeLa cells) that are derived from primary cultures.
- Healthy primary cells are transformed to make them immortal through genetic manipulation.
- Cells from prolonged cultures can be transformed (phenotypically) spontaneously when they obtain multiple ill-defined mutations.
14.To generate immortal cell lines, we target processes that regulate cellular growth and ageing , give some examples of this?
. tumour suppressor genes (p53, pRb)
or
the telomerase enzyme
Some cells require both
15.What does the telomerase enzyme do?
elongates telomeres e.g., in cancerous cells, stem cells, gametes etc
oAs cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (p53,pRb).
16.How can we inhibit the function of tumour suppressor proteins or introduce telomerase to make them immortal?
By taking advantage of viral oncoproteins.
17.How does Simian Virus-40 (SV40) affect P53 and pRb?
• SV40’s T-antigen interacts with the binding domains of p53 and pRb to cause increased growth (can’t bind to DNA binding regions) without losing the function of these proteins.
18.What viral oncoproteins does the Human papilloma Virus (HPV) produce?
E6 and E7
19.How does the viral oncoproteins E6 and E7 affect P53 and pRb?
E6 targets p53 for degradation, and E7 binds to pRb inactivating it- these cells maintain a differentiated phenotype.
20 . How can the telomerase gene can be introduced into a target primary cell ?
-Using transfection methods
o This is done by designing a plasmid vector that contains the gene for selection and a growth promoting gene (telomerase).
o This is transfected into the primary cells which are allowed to grow.
o A selection pressure e.g. antibiotic is added, colonies are formed from bacteria that have taken up the genes. These are selected for culturing.
- What are 6 advantages of cell lines?
- Good growth characteristics. Standard media
- Phenotypic stability
- Defined population
- Molecular manipulation readily achieved
- Good reproducibility
- Good model for basic science
- .What are 5 disadvantages of cell lines?
- Often lose differentiated function
- Cell-substrate interactions dominate
- Does not mimic real tumour conditions
- Lacks cells heterogeneity
- Phenotype needs to be validated
23.. To grow cells in a culture there are conditions and requirements, list these?
o Handled under aseptic conditions (ethanol sanitised surface, gloves).
o Grown on tissue culture treated plastic flasks/dishes.
o Maintained in a warm (37°C) humidified atmosphere (5% CO2).
o In ideal supplemented medium (growth medium RPM 11640 or DMEM) that needs to be replaced by a fresh one every 2/3 days due to nutrient depletion and the collection of debris/waste.
- 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
- For the following features/characteristic, explain how ADHERENT cells would act in that situation:
- Anchorage dependency
- Agitation
- Trypsinization
- Tissue culture treated vessels
- Yield
- Growth limited
- Types of cells
Anchorage dependency =Anchorage-dependent
Agitation=Not required
Trypsinization=Required
Tissue culture treated vessels=Required
Yield=Low
Growth limited=By the surface area
Types of cells=Most types of cell lines and primary cultures
27 . For the following features/characteristic, explain how SUSPENSION cells would act in that situation:
- Anchorage dependency
- Agitation
- Trypsinization
- Tissue culture treated vessels
- Yield
- Growth limited
- Types of cells
Anchorage dependency = Anchorage independent
Agitation= Continous agitation required
Trypsinization= Not required
Tissue culture treated vessels = Not required
Yield= High
Growth limited= By the conc of cells in the medium
Types of cells= Some non-adhesive cell lines such as hematopoietic
- Do E6/ E7 and telomerase transformations result in cell lines with a differentiated phenotype??????
yeaaaaahhh
- When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Bacteria?
pH change
Cloudiness/Turbidity
Precipitation
Stink
30.When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Yeast?
Cloudiness
pH change
31.When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Fungus?
Spores furry growths
pH Change
32.When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Mycoplasma?
Often Covert
Poor Cell adherent
Reduced Cell Growth
- What are Mycoplasma?
Parasitic bacteria lacking cell walls
- When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Virus?
Sometimes Cytopathic (structural changes in host cells that are caused by viral invasion)
- One type of cell culture contamination , is when you have cell line cross-contamination. Why is this most likely to occur?
- Poor tissue culture technique
- Culture of multiple cell lines at one time
- Accidental mixing of cell lines
- List four new in vitro models?
- 2D cell culture
- 3D cell culture
- 3D Spheroid Culture
- 3D organic Culture
- What is a medium pH indicator?
Phenol Red
- When using Phenol red.What would the colours be for a basic, neutral and acidic medium and the correlating pH’s?
pH of 7.4 - 7.6 is BASIC , red/purple
pH of 7 - NEUTRAL , Tomato Red
pH of 6.8 = ACIDIC , Yellow
39 . Please compare Organoid (O) vs Spheroid 3D Culture (S) :
- Where are Each derived from?
O= Derived from stem cells S= Derived from cell line monoculture
- please compare Organoid (O) vs Spheroid 3D Culture (S) :
- How many Cell lineages?
O = Multiple Cell lineages S= Represent Single/Partial Tissue components
- please compare Organoid (O) vs Spheroid 3D Culture (S)
- How do they resemble actual physiology?
O = Recapitulate organ physiological Parameters S= Transiently (briefly) resemble cell organisation
42 . Please compare Organoid (O) vs Spheroid 3D Culture (S) :
-Is the culture long term?
O = Long term Culture S= Difficult to maintain long term
- How can we use patient derived Organoids to study cancer drug resistance?
- Organoids are taken from tumour biopsies are taken and studies before and after treatment.
- Study new drug targets, drug resistance (mutational analysis and survival analysis), histology and optical metabolic imaging.
- What are four ADVANTAGES of Organoids?
o Gene expression as in vivo (87% phenotype and genotype similarity)
o Cells-cell communication is re-established
o Cells are orientated in same ways as tissue
o Ideal platform for individualised therapeutic screening.
- What are four DISADVANTAGES of Organoids?
o Limited amount of tissue in some cases (e.g. prostate).
o Organoids in the same culture are heterogeneous
o Absence of immune cells in culture system
o 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 are 6 chemical methods to transfection?
lipofection calcium phosphate cationic polymer DEAE-Dextran Magnet-mediated transfection Activated dendrimers.
- What is the viral method of transfection?
Infection
- What are 5 physical methods of transfection?
Electroporation
Nucleofection
Microinjection
Biolistic Particle Delivery Laser-faction/Optoinjection
- What are three benefits of using a cationic lipid transfection system?
- Fast
- Simple
- Reproducible
- What is the method of lipofection?
o The positively charged lipoplexes (synthetic nuclear acid carriers with cationic phospholipids) interacts with the negatively charged cell membrane and is taken up by endocytosis.
o It’s then released from the endosome and transported to the nucleus (entry to the nucleus is inefficient – may need mitosis).
o Liposomes as potential drug carriers for drug delivery.
- What is the method of electroporation?
High electric field forms pores in the cell membrane which then reseal (rate of pore sealing is dependent on temperature).
- What is the method of nucleofaction?
o Combination of electroporation and lipofection
o Increased efficiency particularly of non-dividing cells
o Technology is protected under patent
o Different solution and protocols are used for each cell type
- What is the method of viral infection/transduction?
o Exploits the mechanism of viral infection: uses Retrovirus, Adenovirus, but most commonly Lentivirus.
o High transfection efficiency.
o Target cells need to express the viral receptor to work.
o There are safety aspects to consider.
- Summarise this lecture?
o To overcome the short comings of primary cultures, cell lines have been produced
o Cell lines can be spontaneously induced or produced by transfection with viral oncogenes that inhibit tumour suppressor genes or by introducing/over-expressing telomerase.
o Cell transfection can be carry out using chemical, physical or viral methods.
o Rapidly dividing cells can lose differentiated functions → need for different models- Organoids
