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Question 1

What are recombinant proteins?

Answer 1

proteins encoded by recombinant DNA

cloned in an expression vector that supports expression of the gene and translation of messenger RNA
eg. plasmids

Question 2

Recombinant protein expression

Answer 2

1) Choice of cell line - CHOK1 mammalian cells.
2) Transfection - Introducing foreign nucleic acids (DNA,RNA) into the host eukaryotic cell.
3) Selection/screening of suitable cell line.
4) Single cell cloning.
5) Expansion of selected clones.
6) Cell banking

Question 3

4 types of cell banking

Answer 3

1) Research cell bank (RCB)
2) Master cell bank (MCB)
3) Working cell bank (WCB) - commercial use
4) End of production cell bank (EoPCB)

Question 4

Aim of aseptic techniques and sterilization

Answer 4

To prevent contamination from microorganisms and cross contamination with other cell lines.

Question 5

Reagents & media preparation

Answer 5

• Sterilise heat-stable reagents by autoclaving
• Filter-sterilise heat-labile and non-autoclavable media
• Never use same bottle of medium for different cell lines.

Question 6

Laminar flow hood

Answer 6

• reduces risk of introducing contaminants to cultures
• provides a clean working environment but not 100% sterile

Question 7

Types of flow hood

Answer 7

1) Vertical flow hood
2) Horizontal flow hood

Question 8

Vertical Flow hood

Answer 8

Draws in air through the front of the working area into the vent system at the front of the working surface.
80% of this air is recirculated and the remainder expelled.

• More protection to the operator
• Class II and III vertical flow should be used when handling hazardous material

Question 9

Horizontal flow hood

Answer 9

Draws in air from above, filters the air, then forces the air out the front of the working area of the hood

• For media preparation, or when handling non-primate cell lines, culture manipulations can be conducted in horizontal hood.
• Does not offer operator protection against pathogens

Question 10

Advantages of Vertical flow hood

Answer 10

• Hood not as deep, less floor space
• Suitable for compounding sterile procducts
• Safety, air not blowing at operator and sash provides a barrier in front of operator’s face
• Filter on top, easier to access
• Less cross-contamination of items positioned on the work surface

Question 11

Disadvantages of vertical flow hood

Answer 11

• Overhead clearance required for filter servicing
• Cannot place items or hands on top of other items as they may obstruct flow
• Increased turbulent effect of air strike work surface.

Question 12

Advantages of horizontal flow hood

Answer 12

• Reduced turbulent effect on air striking work surface
• No sash, easier to work and position equipment
• Hands and gloves are generally less contaminating since they are downstream the sample

Question 13

Disadvantages of horizontal flow hood

Answer 13

• Filter change requires rear access
• Large samples obstruct laminar air flow
• Blows flumes and/or powders in operator’s face

Question 14

Organizing laminar flow hood for routine work

Answer 14

1) Wipe down hood and surfaces of flasks, trays, bottles etc. with 70% ethanol to remove dust particles and other contaminants.
2) Minimise clutter. Only reagents and apparatus for the work at hand should be in the flow hood.
- Don’t overcrowd the hood, always leave rear vents as clear as possible for best airflow.
3) Position items for maximum efficiency and reduce human traffic.
- Do not cross-over reagents/media/cultures.
4) Cultures should be the last items to be introduced into a hood when working in it.

Question 15

Main contaminants of mammalian cell cultures

Answer 15

1) Bacteria
2) Fungi
3) Mycoplasma

Question 16

What are the effects of contaminants to cell culture?

Answer 16

Contaminants will flourish in the cell culture environment and will rapidly overgrow, and in many cases kill the animal cells by release of toxins, depletion of nutrient medium or depressing the medium pH.

Question 17

Sources of contamination

Answer 17

Techniques, Hygiene, Materials&Reagents, Equipment&Facilities and Imported Biological Materials.

Question 18

How to monitor for contamination?

Answer 18

1) Check by eye and with a microscope (100x-400x) at every handling
2) Check for mycoplasma
3) If culture becomes cloudy overnight and changes from red to yellow, it is contaminated

Question 19

Characteristic features of microbial contamination

Answer 19

1) Sudden change in pH
- Medium colour red to yellow
2) Cloudy media
3) Under low power microscopy (~100x)
- Bacterial: spaces between cells will appear granular or you may see very small black dots. There may be mobility of the bacteria
- Fungi: thin filamentous mycelia and sometimes denser clumps of spores

Question 20

Mycoplasma contamination

Answer 20

-Not visible by naked eye other than through signs of deterioration in culture
- Grows slowly and does not destroy host cells but interfere with rate of cell growth

Question 21

Test to identify mycoplasma contamination

Answer 21

• Fluorescent staining
• PCR
• ELISA
• Immunostaining
• Microbiological assays

Question 22

How to minimise contamination?

Answer 22

• Checking cultures daily by eye and a phase contrast microscope
• Reagents are checked for stability before use
• Ensuring that media are not shared between different users and cell lines
• Ensuring that the standard of sterile technique is kept high at all times.

Question 23

Procedure when contamination occurs

Answer 23

RECORD the nature of contamination and its specificity
DISCARD all contaminated cultures, including media, flask, pipettes and disposable articles that has been used in conjunction with the cultures.
SWAB the laminar flow and work surface with 70%-75% alcohol
REVIEW all sterilisation procedures

Question 24

When should subculture be started?

Answer 24

• All of the available substrata is occupied
• When the cell concentration (cells/mL) exceeds capacity of medium

Question 25

Why should subcultures be started?

Answer 25

In the 2 situations, cell growth ceases or is greatly reduced

Question 26

Advantages of subculturing

Answer 26

• Scale-up is easier
• Trypsin treatment is not required
• Whole process is quicker and less traumatic for the cells

Question 27

Cryopreservation

Answer 27

Using low temperatures to preserve structurally intact living cells and tissues

Question 28

Reasons for cryopreservation

Answer 28

1) Genotypic shift due to genetic instability
2) Senescence
3) Transformation
4) Contamination by micro-organism
5) Cross-contamination

Question 29

Why is DMSO or glycerol added to the cell?

Answer 29

To protect the cell from being punctured during the freezing process.

Question 30

Two types of cultures

Answer 30

1) adherent cultures
2) suspension cultures

Question 31

Examples of sources of contamination

Answer 31

• Techniques: Manipulations, pipetting, dispensing
• Hygiene: Work surface, operator hair, hands, breath, clothing etc.
• Materials & Reagents: Solutions, glassware & screw caps, instruments & pipettes, culture flasks & media bottles
• Equipment & Facilities: Room air, laminar flow hoods, incubators, refrigerators, infestations
• Imported biological materials: Tissue samples and cell lines