Cell Culture Techniques Flashcards
What is a cell culture?
Group of cells grown in a nutrient solution from a single original cell
How can we isolate different cells? (summary)
1) Blood
- Density centrifugation (used to isolate different blood cell populations)
- Immunopurification and FACS (used to isolare specific cells)
2) From solid tissues
- Mechnical and enzymatic disruption to disrupt cells from solid tissue then magnetic immuno-purification technique used to extract cells of interest
- Mechanical and enzymatic disruption not required in explant culture where chondrocytes will migrate away from cartilage
How do we isolate cell populations in blood?
Density Centrifugation
- This takes advantage of the differential density of the cell population and the density gradient medium that is used.
- The type of cell isolated will depend on the density gradient medium used
What do the layers look like following centrifugation?
- Granulocytes and erythrocytes are denser than mononuclear cells and will sedimeny through the density gradient medium
- Mononuclear cells will remain in the top layer
- Lymphocytes will remain in the buffy coat, these can be extracted through isolation of this specific coat
Techniques used to isolate specific cells from blood
- Immuno-purification
- Fluorescence Activated Cell Sorter (FACS)
How does immuno-purification work?
- Coat magnetic beads with an antibody which will bind to a specific cell surface receptor/ antigen present in the cell or interest
- Addition of magnetic field we can extract cells of interest
Fluorescence-activated cell sorter (FACS)
Uses antibodies to isolate cells of interest, but is also based on size
How do we isolate cells from solid tissues
- Mechanical and enzymatic disruption is carried out on solid tissues
- Mechanical (use a scalpel or passing tissue through needles) and enzymatic (through trypsin, collagenase)
- Once extraction has been carried out individual cells are extracted using magnetic immuno-purification
What happens in an explant culture?
- In a cartilage explant culture, chondrocytes will migrate away from cartilage explant spontaneously
- The cartilage explant is set on a plate and chondrocytes will isolate by themselves
What are cell lines and why are they used?
Cell lines are immortalised cells that continue to grow and divide indefinitely in vitro for as long as the correct culture conditions are maintained
Used as they pose fewer disadvantages compared to primary cells
List some advantages and disadvantages of using primary cells?
-
Advantages
- Unmodified
- Good for personalised medicine
-
Disadvantages
- Aberrant expression of some genes
- Variable contamination
- Limited
- Short life-span
- Inter patient variation
- Difficult molecular manipulation
- Phenotypic instabillity
Where are cell lines isolated from?
-
Healthy or cancerous tissues
- (e.g HeLa cells) = HeLa cell line include cells derived from cell carcinoma
- Primary cultures
Cell lines derived from primary cultures what can they either do?
- Survive spontaneously by themselves without manipulation
- Be genetically manipulated in order to transform them and make them immortal
How do we make cell lines immortal?
By genetic manipulation of three different proteins that are genetically manipulated in order to produce immortal cells
- p53
- pRB
- Telomerase enzyme
What are p53 and pRB encoded by?
What do they do?
They are encoded by tumour supressor genes
p53 and pRB both maintain genomic stabillity by mediating cell cycle checkpoints
What is the role of telomerase?
- Reverse transcriptase enzyme carrying its own RNA molecule 3′-CCCAAUCCC-5′ which it uses as a template to elongate telomeres
- Prevents shortening of telomeres hence reducing point at reaching Hayflick limit where cell senescence is triggered
What are the only cells with active telomerase?
Stem cells, gametes and cancer cells
How do we create an immortal cell line?
- Inhibit function of p53 and pRB
- Introduce/ over-express telomerase
How do we inhibit the function of p53 and pRB?
To inhibit tumour supressor activity (p53 and pRb) we use viral oncoproteins in SV40 (simian) and HPV which will target tumour supressor proteins.
What is the mechanism of action of SV40 to inhibit tumour suppressor proteins?
- SV40 oncoviral large T antigen will interact with p53 and pRb protein indirectly
- It will interact with the cells protein DNA binding domain to which p53 and pRb bind to
- This prevents interaction of pRB and p53 with these domains
- However there are still levels of p53 and pRb in the cell due to indirect inactivation, they are still functional just unable to bind to their domains
What is the mechanism of action of HPV to inhibit tumour supressor proteins?
HPV = E6 will directly targets p53 for degradation, and E7 binds to pRb inactivating it
Cell lines made using E6/E7 are believed to maintain a differentiated phenotype
What is believed to be the most efficient way of producing cell lines?
- Immortalisation of cell lines has been proved most effective with inactivation of pRb /p53 through E6/E7 and telomerase transformation
- These are believed to result in cell lines with a differentiated phenotype
How is telomerase transfected into primary cells to immortalise them?
- Design a plasmid containing a gene for selection with selection marker e.g Antibiotic resistance marker
- Insert the telomerase sequence containing gene into the plasmid
- Once plasmid construction is completed the primary cell will be transfected with those vectors
- To check if the gene has been transfected a selection pressure is added (Antibiotic resistance to neomycin)
- Only cells with AB resistance and telomerase gene will survive
What are some advanatages of cell lines?
- Good growth characteristics
- Phenotypic stabillity
- Defined population
- Molecular manipulation readily achieved
- Good reproducibillity
- Good model for basic science
What are some disadvantages of cell lines?
Often lose differentiated function
Cell-substrate interactions dominate
Does not mimic real tumour conditions
Lacks cells heterogeniety
Phenotype needs to be validated
What are conditions and requirements for cell growth in culture?
- Must be handled under aseptic conditions
- Grow on tissue culture treated plastic flasks/ dishes with enough space to add corresponding growth factors and supplements to medium
- Maintained in a warm incubator (37 degrees), humidified atmosphere (5% CO2) and a neutral pH (same cond. as human body)
What is the significance of growth medium maintence in cell culture?
- Ideal supplemented medium needs to be replcaed by a fresh one every 2/3 days due to depletion of nutrients in the medium and due to release of waste products into medium after cell metabolism
- We will change/replace the growth medium when universal indicator will change colour
Describe the difference between adherant and suspension cells?
Adherant cells
- Cells which attacj to solid surface + proliferate
- Low yield
- Growth limited by surface area
- Most types of cell lines and primary cultures
Suspension Cells
- Grow suspended (floating) in liquid medium
- High yield
- Some non-adhesive cell lines (e.g haematopoietic)
What are the two ways in which cell cultures can become contaminated?
-
Microbial 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)
-
Cell lines cross-contamination
- Poor tissue culture techniques
- Culture of multiple cell lines at one time
- Accidental mixing of cell lines
What are new in-vitro cell line models and why are they used?
3D cultures
- Spheroid cultures
- Organoid cultures
These mimic cells growing in the body because they are 3D - unlike 2D cell line models
What is the difference between organoid and spherical cultures?
Organoid are derived from stem cells
- Multiple cell lineages
- Recapitulate organ physiological parameters
- Long term culture
Spheroid are derived from immortalised cell lines but are grown in 3D
- Represent single/ partial tissue components
- Transiently resemble cell organisation
- Difficult to maintain long term
Describe a use of organoid cultures
- Allow the study of cancer drug resistance
- We can extract primary cells from a tumour and grow them in a lab in 3D, they will maintain the 3D confirmation and cell-cell contacts
- We can test different drugs and observe how cells will respond
- If we see that the organoid culture responds well to the drug, we will know the patient will respond well to the drug
List some advantages of organoid cultures
- Gene expression as in vivo (87% phenotype and genotype similarity)
- Cell-cell communication restablished
- Cells are orientated in the same way as tissue
- Ideal platform for individualixed therapeutic screening
Limitations of organoid cultures
- 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 the definition of cell transfection?
Process whereby foreign DNA is deliberately introduced into a eukaryotic cells through non-viral methods including chemical and physical methods in the lab
e.g plasmid, CRISPR, Cas9 complex
What is transfection using viral methods called?
Infection
What type of cell transfection is lipofection?
How does it work?
Lipofection (chemical transfection method)
Takes advantage of the cationic nature of liposomesm they are +ve and allow DNA to be introduced
- Lipoplex containing DNA is posotively charged and hence interacts with cell membrane
- It is taken up into the cell by endocytosis
- Inside the cell the endosome will be broken down and DNA plasmid will be released
- This will be transported into the nucleus
- In t he nucleus molecules will be transported into the nucleus of the cell and the cell can express the proteins of interest
What are the uses of liposomes?
Can be used for transfection of drugs, tissue specific antigens and can be attached to the surface of the liposome
This is so the liposome will only go to the specific tissue of interest
Explain how electroporation occurs?
- Addition of electric fields will allow for the opening of pores in the membrane of the cell which allow plasmids to enter the cell
- Once the material has been incorporated by the cell these pores will be resealed
- Resealing is dependant on termperature
What is a disadvantage of electroporation?
It is toxic
What is nucleofection?
Nucleofection
- Combination of electroporation and lipofection whereby DNA is transfected into the nucleus straightaway hence gene expression is carried out rapidly
- Increased efficiency particularly of non-dividing cells
- Technology is protected under patent
- Different solution and protocols are used for each cell type
Briefly explain viral infection
- 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
