Cell Culture Techniques Flashcards
What are the methods of cell isolation from blood?
Density centrifugation
- entire cell populations
Immunopurification / FACS
- specific cell types
How does density centrifugation work?
Density centrifugation takes advantage of the varying densities of the blood cell populations and the density gradient medium (DGM) that we use
Depending on the medium we use, we can isolate different types of cells
Name an example of a density gradient medium used in density centrifugation
We can mix our blood sample with Ficoll (DGM) that has a density of 1.077 g/mL
After centrifugation we can observe different layers
Which blood cell type sediments as the bottom layer?
Granulocytes and erythrocytes are denser than the other cell layers and so sediment through the DGM to the bottom
Which cells are found towards the top of the blood centrifugation?
Less dense mononuclear cells usually remain towards the top in the plasma interface - can isolate and centrifuge to separate the mononuclear cells
What is the buffy coat?
Can also isolate the buffy coat white layer (lymphocytes) containing copies of the germline info
What techniques can be used to isolate more specific cell types?
In order to isolate more specific type of cells we can use other techniques e.g.
- Immuno-purification
- Fluorescence Activated Cell Sorter
Outline the process of immunopurification
- Magnetic beads coated with antibodies - bind to
specific antigen on cell of interest surface - Coated beads mixed with blood sample and only bind
to cell type of interest - By application of magnetic fields, we can instruct beads
bound to cells of interest
How does FACS isolate specific cell types?
uses cell size and antibodies to separate cells of interest
How are cell types differentiated in FACS?
Uses cell surface markers and size to differentiate between cell types
Give an example where cells are isolated from tissues
E.g. Isolation of hematopoietic placental stem cells that can differentiate into any cell lineage
Or placental endothelial cells
What is the difference between cell isolaton from blood and from tissue?
In tissues cells require isolation from solid tissue so need to carry out a mechanical enzymatic disruption
How do we carry out a mechanical enzymatic disruption of cells?
- Scalpels used to disrupt tissue
- Pass tissue through needles to extract individual cells
What other technique is used to disrupt cells alongside mechanical disruption?
Mechanical disruption is usually combined with digestive enzymatic disruption
e.g.
Dispase, trypsin, collagenase
How are specific cell types extracted from tissue after disruption?
We can now apply magnetic immunopurification techniques to extract the cell type of interest
Outline how mechanical enzymatic disruption is used in explant cultures
Easily carried out in chondrocytes as they migrate away from a cartilage explant - doesn’t require any additional techniques, can occur spontaneously
What are the advantages of using primary cell cultures?
Unmodified; carry all genetic info belonging to patients tumour / tissue they’re isolated from
Makes them good for personalised medicine e.g. therapeutic drug assays: assess response of patient to certain drugs based on their genetic profile
Why are primary cell cultures not good for basic science?
However, for in vitro contours for basic science, primary cells aren’t ideal models as they carry other non-functional genes - contribute in contamination (e.g. bacteria from organism extracted form)
What are some disadvantages of primary cell cultures?
Short life span - can’t be handled after a certain no. of times
Can’t carry out reproducible in vitro analysis using other cell lines - primary cell population will have different characteristics to others
Where are cell lines isolated from?
Can be isolated from healthy or cancerous tissues (e.g., HeLa cells)
Where are cell lines derived from?
Spontaneously
from prolonged culture, multiple ill-defined mutations transformed phenotype
Through genetic manipulation
Transformation of healthy primary cells
How are cell lines generated?
To generate cell lines we target processes that regulate cellular growth and ageing
What 3 genes are manipulated to form immortal cells?
P53
pRB
Telomerase enzyme
What are the roles of p53 and pRB?
P53 and pRB are encoded by tumour suppressor genes and maintain cell cycle checkpoints and regulate genomic instability
What are telomeres?
Telomeres are short tandem nucleotide repetitions found at the ends of each chromosome.
What is the function of telomeres?
To maintain stability of chromosomes and prevent fusion with other chromosomes
What is telomerase?
Telomerase is an enzyme that elongates chromosomes
Why does telomerase elongate chromosomes?
Every time DNA is replicated in a cell, DNA pol. is unable to elongate telomere sequences completely ∴ in each cell division there is telomere shortening
How does certain no. of cell divisions lead to apoptosis?
When cell division no. reaches its limit, telomeres are very short in length causing chromosomal damage
⇒ activates p53 and pRB; lead cell to apoptosis due to damage signal
Which cells contain active telomerase?
Other types of cells also contain active telomerase e.g.
- Gametes
- Stem cells
- Cancer cells
What is the advantage of activated telomerase?
By activating TERT (telomerase enzyme) we can create immortal cells
How can we produce immortal cells?
We can inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions by taking advantage of viral ‘oncoproteins
Which viruses do we use to produce immortal cells?
can use SV40 and HPV to manipulate telomerase enzymes in their viral oncoproteins.
How is SV40 used to produce immortal cells?
SV40’s large T-antigen DNA binding domains interact with p53 and pRb preventing them from binding to E6 and E7 domains. This can cause increased growth without loss of function of these proteins
How are HPV manipulated to produce immortal cells?
E6 in HPV targets p53 for degradation, and E7 binds to pRb inactivating it
Describe the phenotype of viral oncoprotein manipulated cell lines
E6/ E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype
Outline how the telomerase gene is introduced to immortalise primary cells
- Plasmid vector designed carrying gene for selection
marker e.g. antibiotic resistance marker - Insert into plasmid, the telomerase gene sequence we
want to transfect into cells - Completed plasmid vector transfected
- Positively transfected cells identified via antibiotic
resistance marker - Only positively transfected cells show antibiotic
resistance as they have antibiotic resistance gene
Why are cell lines a good model for basic science?
Cell lines show good reproducibility as different labs using the same cell line can obtain the same results from an assay
> makes cell lines a very good model for basic science
Why are cell lines an unreliable source?
Cell lines usually lose differentiated function and revert to become stem cell like without a differentiated phenotype
What are the disadvantages of cell line use?
Show bias to cell substrates in their actions; acquire polarity due to attachments to cell surfaces
Phenotype needs to be validated by an external source - identify cross contaminations, acquired mutations etc.
Outline the conditions and requirements of growth in culture
a) Handled under aseptic conditions
b) Grown on tissue culture treated plastic flasks/dishes
c) Maintained in warm (37°C) humidified atmosphere (5%
CO2) - mimics internal human conditions
d) In ideal supplemented medium that is replaced by fresh
one every 2/3 days*
What conditions need to be maintained for cell culture growth?
Need to ensure cells are grown at optimum pH (7.0) and temperature (37.0c)
Ensure there is enough space to grow in the dish
We need to add the corresponding growth factors and supplements to the medium
What is the consequence of a lack of any of the conditions required for cell culture growth?
If the cells lack any of the aforementioned requirements they will arrest in the cell cycle ⇒ quiescent cells
What are quiescent cells?
Quiescent cells - still alive, but won’t divide and proliferate
Give examples of 2 common growth culture mediums
Growth medium RPMI 1640
Growth Medium DMCM
Why does the growth medium need to be replaced so often?
Medium needs to be replaced every 2-3 days to replenish nutrients and metabolite excretion
What is phenol red?
Phenol red is a medium pH indicator
What causes a colour change in phenol red?
Colour change based on medium pH dependent on metabolite presence
Ideal colour - red (neutral)
What are the two ways culture cells can grow?
Cells can either grow in suspension or attached to a surface
Outline the features of adherent cells
- Grow attached to solid surface
- Anchorage-dependent
- Trypsinization required
- Tissue culture treated vessels
- Low yield
- Growth limited by SA
- Most cell lines and cultures
Outline features of suspension cells
- Grow suspended in a liquid medium
- Anchorage-independent
- Continuous agitation needed
- High yield
- Growth limited by [cells] in medium
- Some non-adhesive cell lines
What are some of the microbial contaminations found in cell cultures?
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 the types of cross contaminations that can occur in cell cultures?
- Poor tissue culture technique
- Culture of multiple cell lines at one time
- Accidental mixing of cell lines
What is the purpose of new in vitro models?
Used to overcome cell line disadvantages
What are the 2 types of 3D cell culture models?
- Organoid
- Spheroid
How are spheroid cells made?
Spheroids are made from established immortalised cell lines that normally grow in 2D but are grown in 3D
What are organoid cells?
Organoids are 3D cultures derived from stem cells normally present in normal tissues
Why are organoids and spheroids better than 2D models?
Organoids and spheroids are better models than the 2D cell cultures, as they replicate 3D growth as in humans - no bias to cell attachments
What areas in cell culturing are spheroids and organoids respectively used for?
Spheroids good for in vitro model = reproducible
Organoids good for drug resistance studies
Outline the features of organoid cells
- Derived from stem cells
- Multiple cell lineages
- Recapitulate organ physiological parameters
- long term culture
Outline the features of spheroid cells
- Derived from cell line monoculture
- Represent single / partial tissue components
- transiently resemble cell organisation
- Difficult to maintain long term
Explain how patient derived organoids are used to study cancer drug resistance
- Extraction of primary cells from a tumour
- Grown in 3D in the lab
- Allows tumour to maintain its 3D structure and cell-cell
contacts - can test different drugs and their efficacy and response -
simulates body well
What are the advantages of organoid use?
- 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 the limitations of organoid use?
- 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?
Process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods (chemical and physical methods in the lab)
e.g. a plasmid, a CRISPR/Cas9 complex
Give examples of chemical methods of transfection
Lipofection Calcium phosphate Cationic polymer DEAE-Dextran Magnet-mediated transfection Dendrimers
Outline the physical transfection methods
Electroporation Nucleofection Microinjection Biolistic Particle Delivery Laserfection/ opto injection
Explain how lipofection occurs
Lipofection takes advantage of the cationic nature of liposomes
DNA by nature is -ve charged - can introduce DNA into those liposomes allowing it to fuse
What are liposomes?
small vesicles surrounded with phospholipid bilayer with positive heads
Outline the mechanism of lipofection
Lipoplexes positively charged
Membrane is negatively charged
- Interaction with the cell membrane
- Taken up by endocytosis
- Release from the endosome
- Transport to the nucleus
- Entry to the nucleus inefficient and may need mitosis
When is lipofection commonly used?
Lipofection significantly used in transfection of drugs
How can we make liposomes tissue specific?
Can make liposomes tissue specific by attaching antigens to their surface
What is the disadvantage of lipofection?
Lipofection however can be quite toxic for the cells
What is the process of electroporation?
Application of electric fields enables us to open pores in the cell membranes making them permeable to the plasmids
Once the plasmid is in the cell, the membrane pores reseal
What needs to occur in electroporation for transcription to occur?
In order to activate the transcription machinery, the cell needs to incorporate the plasmid in its nuclear DNA
What is nucleofection?
Combination of electroporation and lipofection
Outline the features of nucleofection?
Increased efficiency particularly of non-dividing cells
Technology is protected under patent
Different solution and protocols are used for each cell type
How effective is viral infection as a transfection method?
High transfection efficiency
How is viral infection manipulated for transfection?
Package viral components into plasmid and infect other cells
Material release containing both viral components in the supernatant
Name examples of viruses used to transfect cells
Retrovirus, Adenovirus, but most commonly Lentivirus are used
What are the limitations of viral infection as a transfection method?
Target cells need to express the viral receptor to work
There are safety aspects to consider
