Methods for growing cells

1. Methods for growing cells

2. Conditions Necessary for Growing Cells • Appropriate nutrients (Media) • Appropriate Environmental Conditions (pH, Temp) • Adequate Oxygen Supply • Aseptic Conditions

3. 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

7. 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)

9. 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)

10. Starting a Cell Culture • Vial- aliquot of cells of specific cell line is stored frozen in a small glass or plastic vial (usually 1mL)

11. T-Flasks and Roller Bottles • 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.

13. 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

14. 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.