Flashcards in Chapter 2 - Cell Culture Requirements Deck (21)
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
What are the essential EQUIPMENT needed for animal cell cultures?
Cabinet (flow hood)
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
What is laminar airflow?
Airflow in a confined area that moves in a uniform velocity along parallel flow lines
Draw Horizontal, Laminar-flow (clean bench)
See page 7
Draw Verticle, Laminar-flow (safety cabinat)
See page 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.
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.
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)
What are the gaseous compositions in an incubator?
5% CO2 and 95% air
What percent of gas does oxygen account for in an incubator?
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
What is complete media?
Complete media is the combination of basal medium, serum and antibiotics
What components are in basal media and give a brief description of their function(s)?
What are the roles of serum?
Supplies low-molecular-weight nutrients
Supplies carrier proteins for low-molecular-weight nutrients and for water insoluble substances
Polypeptide growth and attachment factors
Detoxifies by binding toxic metals and pyrogens
Acts as a pH buffer
Protects labile essential nutrients
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.
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
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)
What are the requirements of antibodies in cell cultures?
Must eliminate the contaminate
Must not inhibit growth and metabolism of cells in culture
What are the commonly used antibiotics in cell culturing?
Penicillin - inhibits cell wall synthesis
Streptomycin / Gentamycin - inhibits protein synthesis
Amphotericin B - fungi and yeast