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.