methods for growing cells
DESCRIPTION
Methods for growing cells. Conditions Necessary for Growing Cells. Appropriate nutrients (Media) Appropriate Environmental Conditions (pH, Temp) Adequate Oxygen Supply Aseptic Conditions. Small Scale Bacterial Cultures. Agar Plates (for isolating and maintaining pure culture colonies): - PowerPoint PPT PresentationTRANSCRIPT
METHODS FOR GROWING CELLS
CONDITIONS NECESSARY FOR GROWING CELLS Appropriate nutrients (Media) Appropriate Environmental Conditions (pH,
Temp) Adequate Oxygen Supply Aseptic Conditions
SMALL SCALE BACTERIAL CULTURES Agar Plates (for isolating and maintaining
pure culture colonies): Nutrients provided by agar media Temperature controlled by incubator Oxygen supplied by ambient air Aseptic conditions created by autoclaving agar
media and using pre-sterilized petri dishes Aseptic conditions maintained by working in
Laminar Flow Hoods
SMALL SCALE BACTERIAL CULTURES Broth Cultures (for small scale production
and inoculum development for larger cultures): Nutrients provided by liquid media (e.g. LB
broth) Temperature and oxygenation maintained by
shake flasks in incubator shakers (50 mL to 1 L) Aseptic conditions created by autoclaving media
in shaker flasks Aseptic conditions maintained by inoculating
shake flasks in Laminar Flow Hoods and then keeping shake flasks covered by sterile caps/ cotton plugs/ other plugs that are microporous)
SMALL SCALE CELL CULTURE What is needed for healthy cell growth?
Nutrients (media: serum, hydrolysates, undefined, defined)
Temperature (34 to 37C) Oxygen (dissolved oxygen, sufficient for metabolism) pH (~ 6.6 to 7.2; usually a bicarbonate buffering
system) Osmolality (physiologic range, usually near 300
mosm) Growth factors (via inoc. density, carryover, media)
STARTING A CELL CULTURE Vial- aliquot of cells of specific cell line is stored
frozen in a small glass or plastic vial (usually 1mL)
• T-Flask- flat sided plastic flask used for small scale culturing (usually 100-600 mL).
• Roller Bottle- plastic bottle similar to a T-flask, where the cells grow on the surface of the bottle while the bottle rotates media around the bottle (typically 100-400 mL).
• Cells adhere to surface of flask by “treating” the plastic surface. This is usually done by generating highly charged oxygen ions which graft to the plastic, creating a hydrophilic, negatively charged surface which attracts most cell lines. Other methods use chemical coatings, such as poly-D-lysine.
T-FLASKS AND ROLLER BOTTLES
LARGER SCALE CELL CULTURE: SPINNERS
For use with suspension cultures
Advantages Reusable (cost efficient) Scaleable
Disadvantages Variability from vessel to vessel Gassing issues (seals, gas exchange) Impellor shear force Needs to be cleaned
LARGE SCALE BACTERIAL & CELL CULTURES Use Bioreactors:
Nutrients provided by liquid media Temperature, pH, and oxygen controlled using
automatic controller Oxygen provided by bubbling air/oxygen through
a sparger at the bottom of the vessel and stirring the media
Aseptic conditions created by autoclaving the bioreactor (for small bioreactors) or steaming the bioreactor in-place (for larger bioreactors > 10 L)
Aseptic conditions maintained by keeping bioreactor closed at all times and introducing oxygen and nutrients through filters or aseptic connections.