Chapter 2 - Cell Culture Requirements Flashcards Preview

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Flashcards in Chapter 2 - Cell Culture Requirements Deck (21)
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1

What is needed in order to culture animal cells?

Sterile environment to manipulate cultures

Proper culture environment to support cell growth

Nutrient environment to support cell growth

Appropriate physical support

2

What are the essential EQUIPMENT needed for animal cell cultures?

Cabinet (flow hood)

Incubator

Centrifuge

Microscope

3

What does HEPA stand for and what is it?

High Efficiency Particulate Air filter - composed of fiberglass strands with 99.99% efficiency; filtering out particles 0.3um or larger

4

What is laminar airflow?

Airflow in a confined area that moves in a uniform velocity along parallel flow lines

5

Draw Horizontal, Laminar-flow (clean bench)

See page 7

6

Draw Verticle, Laminar-flow (safety cabinat)

See page 7

7

Describe the Forced Air Incubator and list some problems associated

The temperature is controlled by a heater underneath the chamber and water reservoirs and fans control the humidity of the chamber.

The constant blowing of the fans cause accelerated media dessication and increases the changes of cross contamination.

8

What is the Water-Jacketed Incubator? And what advantages does it have over other incubators?

Water-jacketed incubator is surrounded by a layer of water that warms the incubator.

It provides better temperature stability, decreased risk of cross contamination and media desiccation and is much easier to maintain.

9

What roles does the CO2 play in culturing cells?

The CO2 is used in biosynthetic reactions and buffering medium (with bicarbonate to maintain pH at 7.0-7.4)

10

What are the gaseous compositions in an incubator?

5% CO2 and 95% air

11

What percent of gas does oxygen account for in an incubator?

19.9%

12

How is proper gas level achieved?

Constant flow - both air and CO2 flow into the chamber continuously at flow rates to achieve desired CO2 levels. The downside being that it is continuous and large amounts of CO2 is wasted.

Automatic CO2 control system - Co2 is injected on demand to reach desired concentration levels; electronic sensors determine when to adjust levels; works for other gases as well O2, N2 or air

13

What is complete media?

Complete media is the combination of basal medium, serum and antibiotics

14

What components are in basal media and give a brief description of their function(s)?

Bulk Ions

Sugars

Trace Elements

Amino Acids

Other Nutrients

Vitamins

Other Components

15

What are the roles of serum?

Supplies low-molecular-weight nutrients

Supplies carrier proteins for low-molecular-weight nutrients and for water insoluble substances

Hormones

Polypeptide growth and attachment factors

Detoxifies by binding toxic metals and pyrogens

Acts as a pH buffer

Protects labile essential nutrients

16

How is serum obtained?

Serum is obtained from the whole blood of baby cows or horses. The blood is allowed to coagulate in order to convert the fibrinogen into fibrin. Fibrin and blood cells are removed through centrifugation and filtration.

17

What are the disadvantages of serum?

Cost - very expensive $350-500 per 500mL of FBS

Variability - variations between batches that may alter cell culture performance

Undefined - can make protein purification difficult

18

What are some alternatives to serum?

Supplements of hormones and growth factors are added to basal medium such as insulin, transferrin and ehtanolamine.

May need to adapt cells to serum free media (ie. passage cell into media containing less and less serum)

19

What are the requirements of antibodies in cell cultures?

Must eliminate the contaminate

Must not inhibit growth and metabolism of cells in culture

20

What are the commonly used antibiotics in cell culturing?

Penicillin - inhibits cell wall synthesis

Streptomycin / Gentamycin - inhibits protein synthesis

Amphotericin B - fungi and yeast

21

What is a common way in which the physical support in which cell cultures grow on is prepared?

Polystyrene is treated with ozone via plasma discharge exposure which gives the polystyrene a net negative charge.