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Clinical Pathology > 2. Cell Culture > Flashcards

2. Cell Culture Flashcards

1
Q

What are recent uses of cell culture?

A
  • Severe burn and stem cells are harvested
  • The stem cells are sprayed onto the burnt skin using a cell spray gun device
  • The cell application heals the burn
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2
Q

What are examples of methods of isolation of cells from blood?

A
  1. Density centrifugation
  2. Fluorescence activated cell sorter (FACS)
  3. Immuno-purification
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3
Q

Expand on density centrifugation

A
  • Gradient formed by centrifugation
  • Cells/sample placed on top of gradient and centrifuged
  • Each subcellular component will move up or down when centrifuged until it reaches a position where its density matches its surroundings and then will move no further - distinct bands formed - most dense at bottom
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4
Q

Expand on FACS

A
  • Label surface of cells with specific marker for particular cell type
  • Laser used to detect which cells are labelled
  • Labelled cells diverted to particular tube (see flow cytometry)
    • Since the fluorescent cells have a positive charge, they can then get sorted into the container with a negative charge
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5
Q

Expand on immuno-purification

A
  • Antibodies attracted to magnetic beads
  • Antibody binds to antigen on cell surface of protein
  • Magnet used to attract proteins with magnetic beads - purified (other unwanted proteins filtered out)
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6
Q

How do you isolate cells from solid tissues?

A

• Mechanical and enzymatic disruption (e.g. collagenase, dispase, trypsin)
- You then use magnetic immune-purification and take out the cells (e.g. placental endothelial cells)
• Can also isolate cells using explant culture – tissue harvested in aseptic manner, often minced, and pieces placed in cell culture dish containing growth media. ~ Over time progenitor cells migrate out of the tissue and onto the surface of the dish. (these cells can then be further expanded and transferred into fresh dishes)

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7
Q

How do you maintain cells in good condition in a culture?

A
  • Isolated and maintained under aseptic conditions – reducing possibility of contamination, protecting cells and individual researching (in case cells from humans are infected)
  • Cells grown on treated plastic – plates positively charged (as cells negative), also contains ECF proteins, growth media
  • Maintained in warm humidified atmosphere
  • Antibiotics also added to prevent contamination
  • Glutamine added for eliminating ammonia
  • Factors important for growth: temperature, pH, nutrients, GFs (via serum or recombinant proteins)
  • The media is changed often to keep the optimal conditions, including change in the pH as a result of the toxic by-products of the culture
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8
Q

What are primary cells? And what are positive and negative aspects of them?

A

• Primary cells are obtained directly from tissue
• Positive aspects: unmodified
• Negative aspects:
- Aberrant expression of genes (genetic errors more likely if cultured for longer)
- Variable contamination
- Poor growth characteristics, esp. human primary cells
- Interpatient variation – variation in responses of cells likely
- Phenotypic instability – e.g. loss of receptors after hours of isolation, etc
- Molecular manipulation difficult

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9
Q

What is the ideal model of primary cells?

A
  • Good growth characteristics
  • Phenotypic stability
  • Defined population – know what cells are
  • Molecular manipulation readily achieved
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10
Q

How do you produce cell lines?

A

• Derive from primary cultures

  • Can be spontaneous:
    • From tumours or prolonged culture – cells tend to be poorly differentiated, since abnormal, something has triggered them to keep dividing
    • Multiple ill-defined mutations – longer we work with culture, more likely to find out mutation
    • Transformed phenotype
  • Or from genetic manipulation:
    • Transformation of healthy primary cells
    • To generate cell lines, we target processes that regulate growth and ageing, examples:
  • P53 - antioncogene (tumour supressor proteins)
  • Retinoblastoma gene (Rb) - antioncogene (tumour supressor proteins)
  • Telomerase
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11
Q

Why target p53 and Rb when generating cell lines?

A

• They are tumour suppressor genes
• Regulate different parts of cell cycle
- P53 has different checkpoints in the cell cycle
- pRb checks if the G1 phase has progressed well and then allows the cell to continue the cycle
• P53 mutated or missing in many cancers
• Many have established that cell lines have lost a functional p53
• Rb inhibits DNA synthesis and arrests cells in G1 of the cycle

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12
Q

What is telomerase?

A
  • A ribonucleoprotein that protects telomere repeat sequences (TTAGGG) at the end of chromosomes
  • Present and active in only germ cells, some adult stem cells and most cancers – not present in somatic cells
  • Telomerase – can make DNA from RNA (a reverse transcriptase)
  • Telomerase is comprised of the subunits; Telomerase reverse transcriptase (abbreviated to TERT, or hTERT in humans) is a catalytic subunit of the enzyme telomerase, which, together with the telomerase RNA component (TERC), comprises the most important unit of the telomerase complex.
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13
Q

How does p53 work in a normal cell?

A
  • Telomeres at ends of DNA and they protect the DNA from accidental breakage during replication – they get shorter every time replication happens
  • There are telomere binding proteins at the end to protect telomere
  • When telomeres get short, the binding protein is lost (when point of senescence is reached)
  • P53 can bind to unprotected chromosomes
  • Activated p53 triggers growth arrest or cell death
  • So without p53, there is not trigger for cell death so the cells continue to proliferate
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14
Q

What are two ways the process of cell death and p53 can be targeted to stimulate cell growth?

A

1) Using viral genes
• Examples:
* t and T-antigen of Simian Virus-40
* E6/7 protein of Human Papilloma virus (HPV)
• T-antigen interacts with normal p53 and Rb – increase growth without loss of function
• HPV – E6 targets p53 for degradation, E7 binds to Rb to inactivate it (HPV more functionally stable)
• Cells made using E6/7 are believed to maintain a differentiated phenotype

2) Telomerase
• Most adult human cells don’t have telomerase that can increase length of telomeres
• Having telomerase will therefore extend lifespan of particular cell – binding protein will remain at ends and no place for p53 to bind ~ some cells need both introduction of the telomerase gene and inactivation of the pRb for “immortalisation”
• Therefore: increase expression of telomerase -> blockage of p53 -> generate cell line.

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15
Q

How we introduce the oncogenes or the telomerase gene into a target primary cell?

A
  1. TRANSFECTION (delivery of DNA or RNA into eukaryotic cells):
    - Use plasmid with gene for selection (gene that allows us to select for cells that have successfully taken up GPG, e.g. enzyme that can break down toxic compound like antibiotics) and growth promoting gene (e.g. large T-antigen, E6/E7)
  2. SELECTION PRESSURE ADDED
    - Toxic compound (e.g. antibiotic) added to culture
    - If cell has taken up gene for selection, it will therefore successfully divide as it can break down toxic compound, unsuccessful cells will die
    - We then know these cells have also successfully taken up GPG
    - Hope is to incorporate growth promoting gene into host’s genome, so every time cell divides it will express these genes
  3. COLONIES SELECTED
    - Colonies will form from cells that have successfully taken up GPG
    - Colonies selected can be grown individually, can then be investigated
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16
Q

What are the hurdles to overcome when producing a cell line?

A
  • Getting DNA into the cells – DNA is toxic, negatively charged
  • Getting cells to stably incorporate DNA once inside
17
Q

What are different methods of transfection?

A 
A) CaPO4 co-precipitation
B) Lipofection 
C) Electroporation
D) Viral transfection/transduction
E) Nucleofection
18
Q

Expand on CaPO4 co-precipitation

A
  • Forms molecule with Ca2+ and DNA irritates cell membrane and reduces negative interaction of 2 negative entities (DNA and membrane) ~ improves efficiency
  • DNA will precipitate with calcium and the cell will take it up by endocytosis
19
Q

Expand on lipofection

A

Cationic lipid transfection systems
• Method of injecting DNA into cell via a liposome (artificially prepared vesicle composed of lipid bilayer)
• Uses positively charged lipids (cationic head group) and combined with negatively charge genetic material net positive charge (so can ‘mask’ negative charge of DNA – more effective transfection)
• Current discussion on whether liposome gets endocytosed or fuses with membrane (or both)
• Endocytosis:
- Positive liposome interacts with cell membrane and is taken up by endocytosis
- DNA released from endosome (as liposome breaks down)
- DNA transported to the nucleus
- Entry to nucleus inefficient (small amount taken up, and even smaller amount stably incorporated)

20
Q

Expand on electroporation

A
  • Plates of capacitor charged
  • High electrical field temporarily forms pores on cell membrane of cell increases permeability
  • Allows DNA to go through
  • Rate of pore resealing is dependent on temperature – maintaining lower temperature after electroporation reduces rate of pore resealing
21
Q

Expand on viral transfection/transduction

A
  • Gene-targeting vectors most commonly used are lentiviruses, as can enter cells not undergoing cell division. Others include retrovirus and adenovirus.
  • Exploits normal mechanisms of viral infection
  • Usually has high transfection efficiency
  • Disadvantage: viruses used can be harmful to humans (biological scientist)
  • Target cells need to express the viral receptor to work
22
Q

Expand on nucleofection

A
  • Combination of electroporation and lipofection
  • Punch holes cell via electroporation DNA enters via liposome (lipofection) and then enters nucleus
  • Increased efficiency (but less than viral transfection, however less hazardous)
  • Technology is protected under patent
  • Different solution and protocols used for each cell type
23
Q

What are disadvantages of using a cell line?

A

• Most important: rapidly dividing cell often lose differentiated function
• Therefore 2 factors of growth and function are at odds in making cell lines
• Ideal solution would be a cell line that divides when you want more and stops when a study of function is required.
• Authentication of cell lines is needed
- Karyoptyping
- STR profiling

24
Q

How do you overcome loss of function of rapidly dividing cells?

A

• In Vivo studies:
- Conditional mutant
- Or e.g. T-antigen mutant of SV40 spontaneous tumour formation
- Allows turning on/off gene as it becomes unstable at different temperatures
• Changing culture conditions
- E.g. 3D culture, providing the ECM or co-culture (2 cell types)
- Good as genes expressed as in vivo, cell-to-cell communication re-established (incorporating different cell types may also help tissue behave in a natural way, e.g. blood vessel cells can see cell interaction and function), cells oriented same ways as tissue
- Helps regain functions we have previously lost

Clinical Pathology (62 decks)

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