Animal Cell Culture

1. Animal Cell Culture Differences between procaryotes and eucaryotes • Cells are negatively charged. • attach to positively charges surfaces • some cells must attach to grow, others not • examples of surfaces: sephadex, collagen • positively charged vesicles containing stuff you would like to put into the cell will attach to cell surfaces and be taken into the cell

2. Cell Components of note… • Endoplasmic reticulum • network of membrane-bound channels • channels: outside is the cytosol, inside are the ribosomes • e.r. responsible for the protein synthesis and post-translational processing

3. Mitochondria: • respiration occurs there -> ATP produced • independent organelles with their own DNA, capable of independent reproduction • two membranes • smooth outer membrane • folded inner membrane called the cristae • Lysosomes: • small organelles, single membrane • contain hydrolytic enzymes: proteases, nucleases and esterases • Golgi body: • collection and secretion of extracellular proteins • direct intracellular protein traffic Peroxisomes: contain peroxidases – hydrolyze H2O2 Glyoxysomes: glyoxalases (glyoxylic acid metabolism)

4. Nucleus: • two nuclear membranes – form a nuclear envelope • nuclear pores – continuity between nucleus and cytoplasm • chromosomes and histones

5. Growth Medium • glucose, glutamine, amino acids, • serum: liquid extracted from blood of offspring removed from freshly-killed pregnant cows. • proteins: cell attachment factors; metal binding proteins; protease inhibitors • peptides: various growth factors • hormones: stimulate growth and nutrient uptake • nutrients • metabolites • minerals

6. Metabolism • Glucose -> pyruvate via glycolysis • Glucose -> biosynthesis via pentose phosphate pathway • Pyruvate -> CO2, H2O, via TCA cycle • Pyruvate -> lactic acid, fatty acids • Glutamine is a carbon and energy source: • Deaminated to glutamate-> other amino acids • Enters TCA cycle for other amino acids, etc. • Animal cells can synthesize glucose from pyruvate via gluconeogenesis pathway • waste: lactate, ammonia • at high levels, these are toxic • challenge for high density cultures

7. Cultivation of Animal Cells • Tissues are removed from animals and transferred to growth medium • Organs -> lung, kidney, etc. (cells grow attached) • These are primary cultures • Cells can be transferred to new flasks once they have grown into a monolayer • Remove cells with a protease – trypsin, collagenase, pronase or EDTA • Wash cells with serum containing medium (centrifuge gently) • Resuspend in growth medium • Plate onto a fresh flask Differentiated mammalian cells are mortal, however, cancer cell lines are immortal. Animal cell lines include: mammal, insect, fish, crustaceans Insect cells are easier to grow. They grow faster and you can use a baculovirus as a vector for genetic engineering. Insect cells may not have post-translational modifications like mammalian cells.

9. The blastocyst has an outer layer of cells and inside the hollow sphere, there is a cluster of cells called the inner cell mass. Outer cell layer placenta and supporting tissues for fetus in uterus Inner cell mass  all other body tissues  pluripotent

10. Umbilical Cord Stem CellsAbundant source of the stem cells that produce blood and immune cells. Adult Stem CellsFound in the brain, bone marrow, blood, skeletal muscle, skin, fat and elsewhere. They are difficult to identify, isolate and grow in culture, and they may not be as versatile as embryonic stem cells. But then again, what is research all about?

11. Commonly used cell lines • Chinese Hamster Ovary cells: CHO cells • HeLa cells • mouse kidney cells • Commonly used medium • nutrients + 5-20% serum ($100-$500 per liter) • Problems with serum • cost • virus – safety issues • extra-cellular proteins • lot-to-lot variation • availability • foaming • Book: serum-free media contains insulin, transferrin, fibronectin, other protein components • Serum-free media can also be protein-free

12. Hybridoma Cells • antibody-producing lymphocytes fused with cancer cells – myeloma • lymphocytes grow slowly and are mortal, hybridoma cells are immortal and produce antibodies Production of antibody fragments by fungi and bacteria See Nyyssonen and Eini; Bio/Technology 1993 vol 11(5) p. 591.

13. Kinetics of growth Similar to bacterial culture There is a difference between attached and suspended cultures Disposable bioreactors Cell growth measured by actual cell counts “Hemocytometer” Stain cells and drop on the slide – count all the white ones Cell growth is measured in days. Production can continue in non-growth conditions – hopefully! Oxygen requirements: .06 - .2 x 10-12 mol O2/h/cell OUR ~ 0.1-0.6 mmol O2/l/hr Compare to bacteria at 10 – 200 mmol/l/hr!

14. Animal Cells are shear sensitive – cannot sparge reactors • cells respond to shear with apoptosis • Fritted metal fittings create very small bubbles • Chemical (e.g. Pluronic F-68) can be added to provide shear protection • Typical kLa of suspension cultures (106 cells/ml) 5 – 25 hr-1 • Bioreactor Considerations for Animal Cell Culture • Microcarriers: sephadex, etc: 70,000 cm2/liter: get ~ 107 cells/ml • cells grow in mono – multi-layers on microcarriers • Hollow Fiber reactors • cells grow on the outside of the tubes, nutrients pass through the tubes • uncontrolled, unmixed environment • get high cell concentrations (eg hybridoma demonstrated at 5 – 50 mg/ml antibody • Stirred-Tank reactors • use pitched blade or other impeller (10 – 30 RPM for stirrers) • Tank and bubble columns are used (especially with cells on multicarriers)

15. Perfusion reactor • simultaneous cell cultivation and product concentration and byproduct removal • sonic separator • Products from Animal Cell Cultures • 1. Immunobiologicals: • monoclonal antibodies • immunobiological regulators • Used for diagnostic assay systems, therapeutics for biological separation systems (affinity chromatography) • Interferon • Virus Vaccines • Hormones: glycosylated peptides (e.g. erythropoetin) • Enzymes: TPA, collagenase,factor VII, factor VIII, factor X