Cell Culture Techniques Flashcards

1
Q

What is the history of cell culture techniques?

A
  1. They isolated frog hearts in saline solutions then added different reagents to test the change in frog hearts beats.
  2. German embryologist; stained it.
  3. Development of cell culture techniques for growing viruses
  4. Jones Salk and his team grow poliovirus in monkey kidney cells.
  5. Henrietta Lachs had a carcinoma and Gey removed these cells and found they lived for a long time. Used for the discovery of polio vaccine and no. of chromosomes in humans.
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2
Q

What does cell isolation depend on?

A

Depends on the cell types/ tissues we want to isolate

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3
Q

Describe the process of isolating cells from the blood

A
  1. Density Centrifugation: Takes advantage of the different cell densities and the density gradient. This will decide the cells we isolate e.g. granulocytes and erythrocytes are denser than the density gradient above so can be extracted.
  2. Immuno-purification: Use an antibody that binds to the cells of interest (coated beads). Magnetic fields used to extract desired beads.
  3. Fluorescence-activated cell sorter (FACS): Uses antibodies to isolate cells of interest but also based on size rather than just surface marker.
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4
Q

Describe the process of isolating cells from the solid tissues

A
  1. Mechanical and enzymatic disruption is used. This can be mechanical such as tearing tissues; staples.
  2. After disruption, if interested in one cell type; then a technique such as immuno-purification can be used.
  3. Explant culture - don’t need to disruption as some cells move automatically.
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5
Q

Positives of primary cells (derived directly from tissues)

A
  • unmodified

- good for personalised medicine: a lot of drug therapies respond better to specific pt cells

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6
Q

Negatives of primary cells (derived directly from tissues)

A
  • Aberrant expression of some genes - unfunctional protein expression.
  • Variable contamination
  • Limited
  • Short life-span
  • Inter patient variation
  • Difficult molecular manipulation
  • Phenotypic instability
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7
Q

Why are cell lines preferred to primary cultures?

A

As you cannot carry out in vitro analysis in the primary cultures that can be reproduced as it will have different characteristics of other cells.

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8
Q

Where are cell lines produced from?

A
  • Isolated from cancerous tissues (e.g. HeLa cells)
  • Derived from primary cultures:
    • > Spontaneously from prolonged culture, multiple ill-defined, mutations, transformed phenotype so no need for manipulation
    • > Through genetic manipulation and transformation of healthy primary cells so manipulated to make them immortal.
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9
Q

How are cell lines generated?

A

To generate cell lines we target processes that regulate cellular growth and aging

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10
Q

What are the 3 different proteins that are genetically manipulated to produce immortal cells?

A
  • p53 and pRB which are tumor suppressors

- Telomerase: Telomeres which are at the end of chromosomes that are at short tandem repeats.

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11
Q

What is the function of pRB and p53?

A

Maintain cell cycle checkpoints and maintain genomic stability

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12
Q

Where are telomerase found and what is their function?

A

Telomerase are found in stem cells, in gametes, and in cancer cells. When the cells divide over time, the telomeres shorten and eventually cell division stops and causes apoptosis

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13
Q

What is the hybrid limit of telomeres?

A

Telomeres reach a hybrid limit which is very short in length that the chromosome telomeres get damage. This activates apoptosis when they are too short.

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14
Q

Why pRB and p53 work in tandem with telomerase?

A

Limiting the function of pRB and p53 means that the cell will not apoptosis leading to generating immortal cells. It will also induce telomerase.

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15
Q

How is telomerase induced? Use SV40 and HPV as examples

A

Taking advantage of viral ‘oncoproteins’ for example in simian virus-40, the viral proteins large T antigen and small t antigen will target p53 and pRb; likewise, human papilloma virus has E6 and E7 that target p53 and pRb.

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16
Q

How do SV40’s T antigens interact with p53 and pRB?

A

This can cause increased growth without loss of function of these proteins. They do not inhibit; they interact with the DNA binding domains preventing the interaction of p53 and pRB. However, there are still levels within the cell.

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17
Q

How do the E6 and E7 oncoprotein in HPV interact with p53 and pRB?

A

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. Inhibits p53 directly and pRB.

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18
Q

How is the cell line activated?

A
  • By introducing the telomerase gene into the target primary cell. Some cells need both introductions of the telomerase gene and inactivation of the pRB/p53 for “immortalisation”.
  • E6/E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype.
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19
Q

What is the process of colony selection?

A
  1. Design a plasmid containing the gene for transplantation
  2. Insert sequence of gene for a sequence of telomerase
  3. Transfect cells with vectors and use antibiotic selection.
  4. Only cells that are resistant to the antibiotic (mycin) contain the gene for telomerase.
20
Q

Positives of cell lines

A
  • Good growth characteristics in standard media
  • Phenotypic stability
  • Defined population
  • Molecular manipulation readily achieved
  • Good reproducibility
  • Good model for basic science
21
Q

Negatives of cell lines

A
  • Often lose differentiated function
  • Cell-substrate interactions dominate
  • Does not mimic real tumour conditions
  • Lacks cells heterogeneity
  • Phenotype needs to be validated -> In cell lines, need 7 to be sent to companies to check -> to check there’s no contamination or mutation
22
Q

What are the conditions and requirements to grow cells in a culture?

A
  1. Handled under aseptic conditions
  2. Grown on tissue culture-treated plastic flasks/dishes
  3. Maintained in a warm (37oC) humidified atmosphere (5% CO2) - same conditions in humans
  4. An ideal supplement medium that needs to be replaced by a fresh one every 2/3 days
23
Q

What are aseptic conditions?

A

Lab coats, gloves, spray all equipment with ethanol

24
Q

What happens if the conditions are not correct?

A

The cells will become quiescence and will not proliferate. It needs to be replaced to maintain the conditions; need more nutrients as they will have been used so also to remove waste.

25
Q

Why is phenol used?

A

Phenol is used as it changes colours dependent on how fresh the medium is.

  • Red to purple = basic (pH 7.4 - 7.6)
  • Fresh medium = tomato red = neutral (pH 7.0)
  • Yellow = acidic medium (pH 6.8)
26
Q

What are adherent cells?

A

Adherent cells are grown attached to solid surfaces. They are anchorage-dependent and require trypsinization. As well as this, the yield is low as they are limited by the surface area. Some examples of adherent cells are those grown in cell lines and primary culture.

27
Q

What are suspension cells?

A

Suspension cells are cells that are grown suspended in a liquid medium. They are anchorage-independent, continuous agitation is required. The yield is high as it is only conditioned by the concentration of cells in the medium. An example of suspension cells is non-adhesive cell lines such as haemopoietic.

28
Q

What are some microbial contaminations?

A
  • 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)
29
Q

What are some cell lines cross-contamination?

A
  • The poor tissue culture technique
  • Culture of multiple cell lines at one time
  • Accidental mixing of cell lines
    Contamination cannot be eliminated but can be contained
30
Q

What are the new invitro models used?

A

There are two main types of 3D models:

  • Organoid
  • Spheroid
31
Q

What are organoid 3D cultures?

A
  • Derived from stem cells
  • Multiple cell lineages
  • Long term culture
  • Reciptulate organ physiological parameters
  • From primary cells
32
Q

What are spheroid 3D cultures?

A
  • Derived from cell line monoculture
  • Represent single/partial tissue components
  • Transiently resemble cell organization
  • Difficult to maintain long term
  • established from immortalized cell lines
33
Q

Why are 3D cultures better than 2D cultures?

A

Both are better than 2D cultures as grown in 3D as they do in the human body. Don’t depend on interactions between vessels and cells.

34
Q

What are patient-derived organoids allow?

A

They allow the study of cancer drug resistance
- If extracting cells from tumours, they will grow in 3D as organoids and can be tested to see how they respond to drugs. See if the drugs will work on patients.

35
Q

Advantages of organoids

A
  • Gene expression as in vivo (87% phenotype and genotype similarity)
  • Cells-cell communication re-established
  • Cells are orientated in the same ways as tissue
  • Ideal platform for individualized therapeutic screening
36
Q

Limitations of organoids

A
  • Limited amount of tissue in some cases (e.g. prostate)
  • Organoids in the same culture are heterogenous
  • Absence of immune cells in the culture system
  • Unable to mimic in vivo growth factor/signalling gradients: mimics about 75% angiogenic characteristics of tumour
37
Q

What is transfection?

A

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

38
Q

Give examples of chemical, physical and viral transfection

A

Chemical transfection = lipofection
Physical transfection = Electroporation and Nucleofection
Viral transfection is just infection

39
Q

Summarise the lipofection method

A
  1. Interaction with the cell membrane
  2. Taken up by endocytosis
  3. Release from the endosome
  4. Transport to the nucleus
  5. Entry to the nucleus inefficient and may need mitosis
40
Q

Why is lipofection useful?

A
  • It is used for the transfection of the drug as it can be incorporated into the nucleus and can express
  • takes advantage of cationic level feature of lysosomes
    • > DNA is negatively charged and can introduce DNA into lysosomes by endocytosis.
    • > Grow and release a molecule of interest into the lysosome
41
Q

Summarise the electroporation method

A
  1. Application of electrical fluids into the cell
  2. Opens pores in the membrane of the cell
  3. Intake material and close the pores
42
Q

What is the rate of pore sealing dependent on in the electroporation method?

A

It is dependent on temperature

43
Q

What are the advantages of the nucleofection method?

A
  • 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
  • Lipofection is toxic for cells
  • Nucleofection is very effective as DNA is transfected directly into the nucleus.
44
Q

What are the advantages of the viral infection method of transfection?

A
  • Exploits the mechanism of viral infection

- High transfection efficiency

45
Q

What are the most commonly used viruses in the viral infection?

A

Retrovirus, Adenovirus but most commonly Lentivirus are used.

46
Q

Why does the viral method require a transfection step?

A
  • To package viral components with the plasmid of interest and infect different cells to those of interest.
  • The cells will express both viral particles