Cell Culture Techniques Flashcards

1
Q

How do we isolate cells from blood?

A
  • density centrifugation
  • fluorescence activated cell sorter (FACS) - flow cytometry
  • immuno-purification
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2
Q

What conditions do you need for growth in culture?

A
  • isolated and maintain under aseptic conditions
  • Cells are grown on treated plastic
  • maintained at optimum temp
  • given nutrients and growth factors via the medium
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3
Q

What are Primary cells? Give positive and negative aspects?

A
  • derived directly from tissue
  • Positive aspects - unmodified
  • Negatives - abberant expression of some genes (may not mimic normal tissue), ethical issues, poor growth characteristics, variable contamination, inter-patient variation, may have to get tissue from diseased body
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4
Q

What is the ideal model?

A
  • good growth characteristics
  • phenotyping stability
  • defined population
  • molecular manipulation readily achieved
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5
Q

How do we obtain cell lines from tissue?

A
  • spontaneous - from tumours or prolonged culture, multiple ill-defined mutations, transformed phenotype (faster cell divides, the less well it works)
  • genetic manipulation
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6
Q

How do we genetically manipulate a cell to produce a cell line

A
  • need to manipulate processes that regulate growth and ageing
  • target different proteins in cells that are responsible for regulating growth
  • p53, Rb, Telomerase
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7
Q

What are p53 and Rb?

A

Tumour suppressor genes

  • normally prevent a cell from dividing inappropriately
  • regulate different parts of the cell cycle
  • p53 binds to the ends of chromosomes when telomeres get too short - triggers cell death/arrest
  • p53 is mutated/missing in many cancers
  • Rb inhibits DNA synthesis and arrests cells in G1
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8
Q

Which Viral genes are commonly used to generate cell lines?

A
  • Simian virus-40 - t and T-antigen (interact with normal p53 and Rb - increased growth without loss of function)
  • HPV - E6/E7 (E6 targets p53 for degradation, E7 binds to Rb)
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9
Q

Transfecting in telomerase

A
  • prevents erosion of telomeres
  • some cells need it to silence Rb for immortalisation
  • E6/E7 and telomerase are believed to maintain a differentiated phenotype
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10
Q

Cell line generation process

A
  • Introduce foreign DNA
  • Plasmid introduced into cell will express growth promoting gene (e.g. T, E6/E7) and gene interested in
  • Gene for selection expresses enzyme that will break down an antibiotic. Some cells expressing that gene will not die
  • Neomycin will kill all the cells that are not taken up and aren’t expressed
  • this will give little colonies which can be separated and grown out
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11
Q

Hurdles to overcome

A
  • getting DNA into the cells

- getting the cells to stably incorportate the DNA when inside

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12
Q

Methods of transfection

A
  • Calcium phosphate co-precipitation (not very efficient)
  • Lipofection - using cationic lipid transfection systems
  • Electroporation - make little holes in membrane through which DNA can diffuse through
  • Viral transfection
  • Nucleofection (mixture of lipofection and electroporation)
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13
Q

Lipofection

A
  • Surround DNA with a lipid coat - changes charge of DNA from -ve to +ve and means it can readily fuse with cell membrane
  • taken up by endocytosis
  • small amount of DNA will migrate to nucleus where it may be incorporated
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14
Q

Electroporation

A

High electric field forms little pores which DNA can diffuse through - reseal afterwards

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15
Q

Viral transfection

A
  • Have a cell that generates viral particles and expresses selection gene and gene of interest - packaged into viral particles (will not replicate until into second cell)
  • Placed into target cells - these are the cells you are transfecting, they express a receptor to which the virus can bind
  • virus puts its DNA into cell -> integrates with cell’s DNA via reverse transciption
  • very efficient
  • significant safety implications as you are using viruses
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16
Q

Nucleofection

A
  • High energy field blows holes in the cell membrane
  • then placed in lipid coat which can get into the cells and be transported to the nucleus
  • patented technology
17
Q

Long term storage

A
  • Cryo-preservation
  • problems - ice crystals can form and disrupt cell membrane- need to freeze slowly and thaw fast - use preservatives to remove some of the water (DMSO)
18
Q

Give a disadvantage of cell lines and how you can fix it?

A
  • Rapidly dividing cells often lose their differentiated function
  • therefore want to try and manipulate cells in a way to retain funciton but to continue growth
  • can use heat in some cases to stop the dividing and regain some functions
  • changing culture conditions such as ECM, co-culture or 3d culture may also help
19
Q

Example of co-culture

A
  • grow VSMCs and Endothelial cells together
  • when they group together, they arrange as if they were in vivo (EC outside and VSMC inside)
  • cell-cell communication is re-established
20
Q

What are the 3 retroviruses we can use in Viral transfection?

A
  • adenovirus
  • Associate adenovirus
  • Lentivirus