Cell Culture Techniques Flashcards
Describe use of skin cell spray
Harvest stem cells for a severe burn
Stem cells sprayed onto burn site
Using cells spray gun device
Cell application heals burn
Describe Immuno-purification
Antibody-coated magnetic beads
Mixed with heterogeneous cells
Isolation of antigen-expressing cells
Describe use of fluorescence activated cell sorters (facs)
Collection process = sample is injected into a stream of sheath fluid that passes through the flow cell and laser intercepts
Stream carries cell through a vibrating nozzle
Disturbance in stream causes it to break into a droplet containing ideally one cell
An electrical charging ring is placed just at the point where the stream breaks into droplets
A charge is placed on the ring based immediately prior to fluorescence intensity being measured, and the opposite charge is trapped on the droplet as it breaks from the stream
The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge
Describe the return of the stream to neutral when using a FACS
In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off
Describe cell isolation from solid tissues
Mechanical and enzymatic disruption using: dispase trypsin collagenase
Then magnetic Immuno-purification
Discuss positives/negatives of primary cells (derived directly from tissues)
+ve:
Unmodified
Good for personalised medicine
-ve: Aberrant expression of some genes Variable contamination Limited Short life-span Inter patient variation Difficult molecular manipulation Phenotypic instability
Describe the methods of cells derived from primary cultures
Spontaneously, from prolonged culture, multiple ill-defined mutationstransformed phenotype
Through genetic manipulation
Transformation of healthy primary cells
What do we target to generate cell lines
- To generate cell lines we target processes that regulate cellular growth and ageing
Effect on telomeres as cells divide
- As cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (p53,pRb)
How can we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions?
Answer: taking advantage of viral ‘oncoproteins’
Virus = Simian Virus-40 (SV40) Viral oncoprotein = Large T antigen Small t antigen Cellular targets = p53 + pRb
Virus = Human Papilloma Virus (HPV)
Viral oncoprotein = E6 + E7
Cellular targets = p53 + pRb
Compare the use of SV40’s T-antigen and E6/E7
V40’s T-antigen interacts with p53 and pRb. This can cause increased growth without loss of function of these proteins
- E6 targets p53 for degradation, and E7 binds to pRb inactivating it
Cell lines made using E6/ E7 oncoproteins are believed to maintain a differentiated phenotype
Describe what is required for immortalisation of telomeres
- The telomerase gene can also be introduced into a target primary cell.
- Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for “immortalisation”
Effect of E6/E7 + telomerase transformations
🡪 E6/ E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype
List positives of cell lines
Good growth characteristics. Standard media Phenotypic stability Defined population Molecular manipulation readily achieved Good reproducibility Good model for basic science
Conditions and requirements for growth in culture
Handled under aseptic conditions
Grown on tissue culture treated plastic flasks/dishes
Maintained in a warm (37°C) humidified atmosphere (5% CO2)
In ideal supplemented medium that needs to be replaced by fresh one every 2/3 days* (*Phenol red is a medium pH indicator)
Adherent vs suspension cells - definition
Adherent first then suspension
Cells that grow attached to a solid surface
Cells that grow suspended (floating) in a liquid medium
Adherent vs suspension cells - Anchorage dependency
Adherent first then suspension
Anchorage-dependent
Anchorage-independent
Adherent vs suspension cells - Agitation
Adherent first then suspension
Not required
Continuous agitation is required
Adherent vs suspension cells - Trypsinization
Adherent first then suspension
Required
Not required
Adherent vs suspension cells - Tissue culture treated vessels required?
Adherent first then suspension
Required
Not required
Adherent vs suspension cells - yield
Adherent first then suspension
Low
High
Adherent vs suspension cells - Growth limited by what
Adherent first then suspension
By the surface area
By the concentration of cells in the medium
Adherent vs suspension cells - Types of cells
Adherent first then suspension
Most types of cell lines and primary cultures
Some non-adhesive cell lines such as hematopoietic
List the microbial contaminations of cell cultures
a) Bacteria (pH change, cloudiness/turbidity, precipitation, stink)
b) Yeast (cloudiness, pH change)
c) Fungus (spores furry growths, pH change)
d) Mycoplasma (often covert, poor cell adherent, reduced cell growth)
e) Virus (sometimes cytopathic)
List the Cell lines cross-contamination
of cell cultures
- Poor tissue culture technique
- Culture of multiple cell lines at one time
- Accidental mixing of cell lines
List the negatives 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
Organoid vs spheroid 3D culture
Organoid first then spheroid
Derived from stem cells vs cell line monoculture
Multiple cell lineages vs represent single/partial tissue components
Recapitulate organ physiological parameters vs transiently resemble cell organization
Long term culture vs difficult to maintain LT
Patient-derived organoids allow what
- Patient-derived organoids allow the study of cancer drug resistance
Organoids advantages
Advantages:
- 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
Organoids disadvantages
Limitations:
- 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
Define 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
Describe Lipofection
Using cationic lipid transfection systems
Lipoplexes positively charged
Membrane is negatively charged
Unilamellar liposomal structure w/plasmid DNA to form a net positive charge
- Interaction with the cellb membrane
- Taken up by endocytosis
- Release from the endosome
- Transport to the nucleus
- Entry to the nucleus inefficient and may need mitosis
Li[psomes - use
Liposomes as potential drug carriers for drug delivery
Electroporation - describe
Use plates of a capacitor
Surrounding cell + plasmid DNA
High electric field forms pores which then reseal
List characteristics of nucleofection
- Combination of electroporation and lipofection
- Increased efficiency particularly of non-dividing cells
- Technology is protected under patent
- Different solution and protocols are used for each cell type
Viral infection/transduction - list characteristics
- 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.
