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(2020-21 BY2BT2) Biotechnology > Applications of mammalian cells > Flashcards

Applications of mammalian cells Flashcards

1
Q

What are antibodies?

A

A naturally occurring protein used by the immune system to identify and neutralize foreign objects (e.g. bacteria, viruses). Each antibody recognizes a specific antigen unique to its target.

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2
Q

What is an antigen?

A

Any substance that causes the immune system to produce antibodies against it.

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3
Q

What is an epitope?

A

The specific region on the antigen that is recognized by the immune cells.

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4
Q

What are monoclonal antibodies?

A

Monoclonal (MAb): identical antibodies, produced by one type of immune cell.

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5
Q

What are polyclonal antibodies?

A

Polyclonal: derived from different cell lines, mixture of antibodies.

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6
Q
A
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7
Q
A
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8
Q

What are polyclonal antibodies produced by?

A

Many B cell clones

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9
Q

What do polyclonal antibodies bind to?

A

Multiple epitopes of all antigens used in the immunisation

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10
Q

What antibody class do polyclonal antibodies belong to?

A

A mixture of different antibody classes (isotypes)

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11
Q

What antigen binding sites do polyclonal antibodies have?

A

Different antigen-binding sites

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12
Q

Are polyclonal antibodies expensive or cheap?

A

Cheaper

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13
Q

Describe the yield of poyclonal antibodies

A

Limited supply

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14
Q

What is the potential for corss reactivity of polyclonal antibodies?

A

High

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15
Q

What are monoclonal antibodies produced by?

A

A single B cell clone

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16
Q

What do monoclonal antibodies bind to?

A

A single epitope of a single antigen

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17
Q

What antibody class do monoclonal antibodies bind to?

A

All of a single class of antibodies

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18
Q

What are the antigen binding sites of monoclonal antibodies?

A

The same antigen binding sites

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19
Q

Are monoclonal antibodies expensive or cheap?

A

Expensive

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20
Q

Describe the yeild of monoclonal antibodies

A

Infinite supply

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21
Q

What is the potential for cross reactivity of monoclonal antibodies?

A

Low

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22
Q

What are the four types of therapeutic monoclonal antibody?

A
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23
Q

Describe chimeric monoclonal antibodies

A
  • Variable domains (VH VL ) from mouse & effector region (CH CL) from human
  • Effector region is human
  • Problems of immune responses
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24
Q

Describe humanised monoclonal antibodies

A
  • CDRs (complementarity determining regions) from mouse & rest from human
  • Effector region is human
25
Q

What are the 2 major funtions of antibodies?

A
  • To recognise and bind to the antigen;
  • To induce immune responses after binding.
26
Q

What are the variable and constant region of antibodies?

A
  • The variable region mediates binding.
  • The affinity for a given antigen is determined by the variable region.
  • The constant region mediates the immune response after binding.
  • Different classes of constant regions generate different isotypes (IgG, IgM, IgA, IgD, IgE).
27
Q

History - milestones

A

1975 – Cesar Milstein and Georges Kohler: ‘The Hybridoma technique’

1984 – Milstein and Kohler received Nobel prize

1986 – First mouse monoclonal antibody approved for human use (Muromonab - CD3 – immunosuppressant for organ transplants)

2003 – First human monoclonal antibody (Adalimumab = Humira – for rheumatoid arthritis)

28
Q

Mab production: The Hybridoma Technique (1)

A
  1. Immunization of a mouse
  2. B cell isolation from spleen
  3. In parallel, the cultivation of myeloma cells (lack the HGPRT gene - hypoxanthine-guanine phosporybosyl transferase)
  4. Myeloma and B cells fusion (electrofusion or chemically)
  5. Culture of the fused cells in HAT medium (hypoxanthine, aminopterin, thymidine) for 10-14 days. Unfused myeloma cells will die in the HAT medium. Unfused B-cells die because they have a short lifespan.
  6. Dilution of the hybridoma suspension until one cell per well.
  7. Cell line screening for antibodies of appropriate specificity (by using ELISA).
  8. in vitro (a) or in vivo (b) multiplication
  9. Harvesting of MAbs
29
Q

The Hybridoma technique (2)

Compare the in vivo method to the in vitro method

A

In vivo method: Injected in mice (in peritoneal cavity, gut), produce tumours containing antibody-rich fluid (ascites fluid).

In vitro method: Grown indefinitely in cell culture media.

30
Q

Compare the properties (price, yield etc) of in vivo and in vitro methods

A
  • In vivo method: is cheaper, higher MAb production yield (5-20 mg/ml), unethical, requires specialised personnel (special licenses for handling animals)
  • In vitro method: ethical, doesn’t require specialised personnel, lower MAb productivity (5-10 µg/ml), some hybridoma lines don’t grow well in culture
31
Q

The Hybridoma technique (3)

A

Microencapsulation of the hybridoma cells in alginate capsules → significantly increased the Mab yields (10-100 µg/ml) as a result of higher cell density

Damon Biotech (Abbott Biotech) Company and Cell-Tech use encapsulated hybridoma cells for large-scale production of MAbs. They employ 100-liter fermenters to yield about 100 g of MAbs in about 2 weeks period.

32
Q

The Hybridoma technology:

A
  • Laborious
  • Expensive
  • Time consuming
  • Often caused immune reactions in patients
33
Q

What is Recombinant DNA technology?

A

Recombinant DNA technology is the insertion of DNA molecules/particles from a different species into a host organism (expression system) to produce useful products.

34
Q

State EXPRESSION VECTORS

A
  • Plasmid DNA
  • Virus vector
  • Bacterial vector
35
Q

State EXPRESSION SYSTEMS

A
  • Prokaryotic – bacteria (e.g. E. coli)
  • Eukaryotic

yeast (e.g. Sacharomyces cerevisiae)

viral (e.g. Baculovirus)

mammalian (e.g. CHO)

36
Q

Expression vectors and systems

A
37
Q

Expression systems: Mammalian cells (e.g. CHO, HEK293)

A
  • Increased level of expression
  • Production of native structure proteins (correctly folded with appropriate bonding)
  • Easy to scale up by fermentation
  • Expensive
  • Slower growing
38
Q

Expression systems: Bacterial cells (e.g. E. coli)

A
  • Simple, well-understood genetics
  • Easy to manipulate genetically
  • Very cheap
  • Fast expression with short doubling time
  • Easy to scale up by fermentation
  • Protein folding issue resulting in insoluble inclusion bodies
  • Low yield for large proteins
  • Glycoprotein modification issue
39
Q

Define transformation

A
  • The alteration of the genetic composition of a cell via the uptake of foreign DNA
  • Naturally in bacteria by conjugation (intra- or inter- species)
  • Naturally in bacteria by transduction (viral- mediated DNA incorporation)
  • Artificially by chemical methods and by electroporation
40
Q

What is transfection?

A

The transformation and infection of cells to introduce foreign DNA.

41
Q

Describe transient transfection

A
  • No genomic integration
  • Short term expression (24-48 h)
  • DNA is degraded by nucleases or diluted during cell division
  • Useful for rapid analysis of phenotype or for recombinant protein production
  • Works best with supercoiled circular plasmids
  • Chemical methods or electroporation
42
Q

Describe stable transfection

A
  • DNA is integrated into the cell genome and is replicated with the host DNA
  • Permanent
  • Sustained expression for long periods of time
  • Works best with linear DNA
  • Viral or microinjection
43
Q

Transfection – What are the chemical methods?

A
  • Calcium phosphate
  • Positively charged polymers
  • Liposome
44
Q

Describe chemical methods of transfection

A
  • Rely on masking the negative charges of the phosphate backbone of DNA to facilitate precipitation/cell uptake
  • Particulate DNA complexes are taken up into cells by endocytosis
  • Naked DNA is digested within minutes in the cytosol by nucleases
  • DNA is delivered to the nucleus by an unknown mechanism
  • Not clear whether DNA is delivered into the nucleus as a particulate (protected) or naked
45
Q

What are the advantages of calcium phosphate transfection?

A
  • very simple
  • Inexpensive
  • Well established
  • Works for most cell types
46
Q

What are the disadvantages of calcium phosphate transfection?

A
  • Works for adherent cells only
  • Skill and experience required to get good precipitates
  • Not particularly efficient in some cell types
  • Many cells do not like the adherent precipitate
  • Difficult to automate or perform as a high throughput method
47
Q

Transfection using positively charged polymers (1)

A
  • DNA adheres to the DEAE-dextran and remains soluble
  • The complex adheres to cells and is taken up by endocytosis
48
Q

What are the advantages of transfection using positively charged polymers?

A
  • Gentle, low toxicity
  • Works well for cells in suspension
  • High volume transfections
49
Q

What are the disadvantages of transfection using positively charged polymers?

A
  • Doesn’t work well in many cell types
  • Doesn’t work well for stable transfection
  • Unclear mechanism of action makes optimisation difficult
  • Moderately expensive
  • Low throughput
50
Q

What are lipsomes?

A

Liposomes are vesicles composed of a lipid bilayer surrounding a hollow core into which drugs or other molecules can be loaded for delivery.

51
Q

What are Unilamellar liposomes?

A

Unilamellar liposomes can be formed from cationic lipids by:

Sonication →uneven liposome size, not reproducible

Microfluidisation →regular small, stable liposomes, reproducible

  • DNA is mixed with the liposomes and binds electrostatically
  • Liposome/DNA complexes enter cells by fusion with the plasma membrane
52
Q

What are the advanatages of iposomes-mediated transfection?

A
  • Very simple to perform and optimize - anyone can do it.
  • Easy to automate, high throughput
  • Reliable and reproducible
  • Stable and transient assays work well
  • Works well with many cell types and also in vivo, either adherent or non-adherent cells
53
Q

What are the disadvanatages of iposomes-mediated transfection?

A

Many formulations require use of serum free or serum reduced medium for good efficiency (e.g. all types that use neutral lipids)

Some formulations are unstable to oxygen

Variable toxicity necessitates careful optimization for many types (e.g. Lipofectamine)

VERY expensive to buy (but almost free to make)

54
Q

State a non-chemical transfection method

A

Electroporation

55
Q

What is elecroporation?

A

Uses an electrical field that transiently permeabilizes the membrane. During this period, the foreigner material can enter the cell.

  • Cells and DNA are mixed and placed into a cuvette between two electrode plates.
  • High DC voltage applied as a pulse.
  • Optimised by altering voltage, pulse length, wave form.
56
Q

What are the advantages of electroporation?

A
  • High transfection efficiency
  • Effective for stable transfection
57
Q

What are the disadvantages of electroporation?

A
  • Only works for cells in suspension
  • Devices for transfecting adherent cells are tricky and cumbersome to clean
  • Requires optimization as cell permeability can lead to cell damage
  • Experienced users are required
  • Very sensitive to salt concentrations
  • Expensive
58
Q

Non-chemical transfection methods-Viral transfection (also known as transduction)

A
  • Used for hard-to-transfect cell types
  • Commonly used in clinical research
  • Often 100% transfection efficiency
  • Viruses have evolved to efficiently introduce nucleic acid into cells
  • Can be used for in vivo transfection
  • Engineered for maximum infection, but replication defective by mutation/deletion of gag, pol, env
59
Q

Give examples of viral transfection (transduction)

A
  • Adenoviruses, retroviruses (lentiviruses) →used extensively for gene delivery in mammalian cell culture
  • Baculovirus → expression in insect cells for producing recombinant glycoproteins or membrane proteins.

(2020-21 BY2BT2) Biotechnology (10 decks)

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