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

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

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 H 2 O 2 Glyoxysomes: glyoxalases (glyoxylic acid metabolism)

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

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

Metabolism Glucose -> pyruvate via glycolysis Glucose -> biosynthesis via pentose phosphate pathway Pyruvate -> CO 2, H 2 O, via TCA cycle Pyruvate -> lactic acid, fatty acids Glutamine is a carbon and energy source: 1.Deaminated to glutamate-> other amino acids 2.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

Cultivation of Animal Cells 1.Tissues are removed from animals and transferred to growth medium 2.Organs -> lung, kidney, etc. (cells grow attached) 3.These are primary cultures 4.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.

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

Umbilical Cord Stem Cells Abundant source of the stem cells that produce blood and immune cells. Adult Stem Cells Found 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?

Commonly used cell lines Chinese Hamster Ovary cells: CHO cells HeLa cells mouse kidney cells Commonly used medium nutrients % 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

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.

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: x mol O 2/h/cell OUR ~ mmol O 2 /l/hr Compare to bacteria at 10 – 200 mmol/l/hr!

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 k L a of suspension cultures (10 6 cells/ml) 5 – 25 hr -1 Bioreactor Considerations for Animal Cell Culture Microcarriers: sephadex, etc: 70,000 cm 2 /liter: get ~ 10 7 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)

Perfusion reactor simultaneous cell cultivation and product concentration and byproduct removal sonic separator Products from Animal Cell Cultures 1. Immunobiologicals: monoclonal antibodies immunobiological regulators a)Used for diagnostic assay systems, therapeutics for biological separation systems (affinity chromatography) b)Interferon 2.Virus Vaccines 3.Hormones: glycosylated peptides (e.g. erythropoetin) 4.Enzymes: TPA, collagenase,factor VII, factor VIII, factor X