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
• Brewers and bakers 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

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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

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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

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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?
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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

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Cell Characteristics

• 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

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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

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Mammalian Disadvantages

• Viruses
• Mycoplasma
• Slow growth rate
• Serum costs
• Risk of genetic instability

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Mammalian Advantages

• Good at making mammalian proteins
• Secretory signals
• Post-translational modifications

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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