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

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”

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