1. Part 2: Choosing a Host Cell
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 Gram positive bacteria
E. coli
Produce lipopolysaccharide (an endotoxin)
Gram positive bacteria
B. subtillus
No endotoxins

5. Industrial Yeast Eukaryotic S. cerevisiae P. pastoris
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 Protection by isolation
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 Various cell types 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. Cell Characteristics Normal mammalian Transformed cells
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”

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