Basics of Cell Culture

1. Basics of Cell Culture Part 2: Choosing a Host Cell

2. Overview • Kinds of cells used in industry • Advantages • Disadvantages • Large scale cell growth issues • Sterility • Cleanliness • Consistency

3. Cells Used in Bioprocessing • Different options • Choice based on many variables • No perfect single choice • Bacterial cells • Yeast cells • Insect cells • Mammalian cells

4. Industrial Bacteria • Gram negative bacteria • E. coli • Produce lipopolysaccharide (an endotoxin) • Gram positive bacteria • B. subtillus • No endotoxins

5. Industrial Yeast • Eukaryotic • S. cerevisiae • Brewer’s and baker’s yeast • Alcohol dehydrogenase system • P. pastoris • Methanol producer • High level of protein synthesis • High cell density

6. Microbial Advantages • Simple media • Fast growth rates • Sturdy cells • Cell walls outside plasma membrane • Faster mixing possible • Faster sparging rate • Long history of large-scale use

7. Microbial Disadvantages • Potentially infectious • Endotoxin (with some cells) • Sturdy cells • Harder to break open • Harder to purify internal protein • Simpler post-translational modifications • Bacterial inclusion bodies

8. Protein Modifications • Functional groups added to proteins • After translation is done • Increase functionality of protein • Membrane association • Extra-cellular protection • Activity regulation • Limited in bacteria, extensive in mammals

9. Inclusion Bodies • Bacterial defense • Normally have balanced protein profile • Too much of one protein is “bad” • Protection by isolation • Excess protein stored in aggregation • Too much for cell to process • Active protein may be recoverable

10. Renaturing Inclusion Bodies • Lyse cells • Separate soluble from particulate • Add strong protein denaturant • Aggregate held by hydrophobic interactions • Denaturant breaks hydrophobic bonds • Renature protein into proper conformation • Optimization can be difficult

11. Mammalian Cells • Various species used • Monkey • Rat • Mouse • Hamster • Various cell types used • Each originally from a single animal

12. Primary Cell Culture • Obtain source of cells (organ, tissue, etc.) • Dissociate cells • Scissors • Digestive enzymes • Grow on treated plates • Isolate cell of interest • Fastest growing cells? • Selective conditions for cell type? • Divide cells as they fill up surface of plates

13. Mammalian Cells- Live or Die?

14. Transformation • Different from bacterial “transformation” • De-differentiation • Anchorage dependence may be lost • Lower serum requirement for growth • Loss of normal behaviors or functions • Immortality • Induced by some chemicals or viruses • Spontaneous in some cell types

15. Normal mammalian Surface attachment* Contact inhibition Require growth stimuli Mortal Non-tumorigenic “Typical behaviors” Transformed cells May not require surface Overgrow monolayer Independent of stimuli Immortal May be tumorigenic Loss of “typical behaviors” Cell Characteristics

16. Mammalian Cell Culture • Mammalian cells used industrially are transformed • Continuous cell lines • Immortal • Usually suspension culture • Chinese Hamster Ovary (CHO) • Surface or suspension growth • Versatile synthetic machinery • Intracellular or extracellular proteins

17. Mammalian Disadvantages • Viruses • Mycoplasma • Slow growth rate • Serum costs • Risk of genetic instability

18. Mammalian Advantages • Good at making mammalian proteins • Secretory signals • Post-translational modifications

19. “Fermentor” vs. “Bioreactor” • Microbial cells grown in “fermentor” • Original stirred tank reactor (STR) • Generally use rapid stirring • Mammalian cells grown in “bioreactor” • Same general structure • Gentler mixing since cells more fragile • Size range more limited

20. Review • Many cell options are available • Choose on cost and usability • Media/growth expense • Ability to make protein properly • Balance advantages vs. disadvantages • Bottom line is useful product • Minimize cost but make product