1. Considerations in Using Animal Cell Culture Structure and biochemistry of animal cells

2. Eukaryotes: Animal cell Animal cells are eukaryotic cells, or cells with a membrane- bound nucleus. contain other membrane-bound organelles, or tiny cellular structures, that carry out specific functions necessary for normal cellular operation.

3. Figure 1: structure of animal cell

4. Structure and function of organelles OrganellesFunction Nucleus Regulate synthesis of proteins in cytoplasm through mRNA Nucleolus- site of ribosome synthesis Chromosome: nuclear material (DNA) Plasma membrane selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells Smooth Endoplasmic reticulum Lipid synthesis Rough endoplasmic reticulum (with ribosome) Critical in protein synthesis and posttranslational processing Mitochondria Site of respiration and production of ATP. Golgi complex Completion of complex glycosylation Collecting and secreting extracellular proteins Directing intracellular protein traffic to other organelles.

5. Lysosomes Contain hydrolytic enzymes (proteases, nucleases and esterases) Digestion of certain food particles ingested by the cell Peroxisomes and glyoxysomes Cytoskeleton Provide cell mechanical strength and control its shape critical in cell movement Transduction of mechanical forces into biological responses Separation of chromosomes into two daughter cells during cell division

6. Characteristic of animal cell Size: between 10-30 μm. Shape: (spherical or ellipsoidal). Cell structure: a)Do not have cell wall. b)Surrounded by thin and fragile plasma membrane. c)Has microvilli- to increase surface area. d)Surface of the cell is negatively charged and cells tend to grow on positively charge surface (for anchorage-dependent cells). e)Posses specific cell surface receptors that adhere to ligand on the surface.

7. Characteristic of animal cell Some animal cells are non-anchorage dependent and grow in suspension culture. Has cytoskeleton or system of protein filaments (actin filaments, intermediate filaments and microtubules)-provide cell mechanical strength, control shape and guide cell movement. Some animal cells contain cilia- used to transport substrate across the cell surface.

8. Typical growth media culture contains glucose, glutamine, non-essential and essential amino acids, serum, mineral salts (e.g: DME, Dulbecco’s Modified Eagle’s media)

9. Methods Used for The Cultivation of Animal Cells

10.  Differ significantly from those used with bacteria, yeast and fungi  Tissue excised from specific organs of animal such as lung and kidney, under aseptic conditions are transfer into a growth medium containing serum and small amount of antibiotics in small T-flasks.  Primary mamalian cells do not normally form aggregate  Grow in the form of monolayer on support surface such as glass surface or flasks.  Using the proteolytic enzyme trypsin, individual cells in a tissue can be separated to form single-cell cultures.

11. Steps in animal cells culture 1)Excised tissues are cut into small pieces (~ 2mm 3 ) 2)Placed in an agitated flask containing dilute solution of trypsin (~0.25% w/v) in buffered saline for 120 min at 37°C 3)The cell suspension is passed through a pre-sterilized filter to clear the solution 4)Cell are washed in the centrifuged 5)Cells are re-suspended in growth medium 6)Placed in T-flasks or roller bottles

12. Anchorage-dependent cells : Cells growing on support surfaces Nonanchorage-dependent cells : Cells grow in suspension culture Primary culture: The cells that directly derived from excised tissues Secondary culture: A cell line obtained from the primary culture

13. 1.Removal solution for cells : EDTA, TRYPSIN, COLLAGENASE OR PRONASE 2.The exposure time for cell removal : 5- 30 min (37°C) 3.After cells are removed from surfaces, serum is added to the culture bottle 4.The serum-containing suspension is centrifuged, washed with buffered isotonic saline solution and used to inoculate secondary culture Step for removing cell

14. Mamalian cells are divided by Normal (mortal) and immortal (continuous/transformed) Normal: Divide only for limited of generation (30generations) Transformed: Can be propagated

15. Characteristic: Contact inhibition: cell division is inhibited when cell’s surface is in contact with other cell No contact inhibition: the cells do not sense the presence of other cells and keep dividing

16. Hybridoma Cell  Obtained by fusing lymphocytes (normal blood cells that make antibodies) with myeloma (cancer) cells  Lymphocytes producing antibodies grow slowly and are mortal  After fusion with myeloma cells, hybridomas become immortal, can reproduce and produce antibodies.

17. Steps in formation of a hybridoma for making antibody (a) Antigen is injected into a mouse (f) The hybrid cell grows well in tissue culture and makes a single monoclonal antibody (e) Myeloma are fused with lymphocytes (c) Lymphocytes are collected from the mouse (b) Lymphocytes in the mouse are activated to produce specific antibodies to the antigen (d) Myeloma (cancer) cells growing in tissue culture are produced

18. Serum  A typical growth medium for mammalian cells contains serum (5-20%), inorganic salts, carbon and energy sources, vitamins, trace elements, growth factor and buffer in water.  Serum is a cell-free liquid recovered from blood (FBS- fetal bovine serum; CS-calf serum; HS-horse serum)  Serum is known to contain amino acids, growth factors, vitamins, certain protein, hormones, lipids and minerals.

19. Serum’s function: 1.To stimulate cell growth and other cell activities by hormone and growth factors 2.To enhance cell attachment by certain proteins such as collagen and fibronectin 3.To provide transport proteins carrying hormones, minerals and lipids

20. Kinetic Growth of Mammalian Cell Culture No.CellsGrowth condition 1.Mammalian cells37°C, pH ~7.3 Doubling time: 12 – 20 h Need to be gently aerated and agitated Buffer used: Carbonate buffer/CO 2 -enriched air/HEPES 2.Insect cells28°C, pH 6.2 3.Fish cells25°C-35°C, pH 7 – 7.5

21.  Similar to microbial growth  Stationary phase is relatively short  Consentration of viable cells drops sharply as a result of toxic accumulation (lactate-from glucose metabolisme and ammonium-from glutamine metabolisme)  Reach peak value from 3 to 5 days  Product formation (monoclonal antibody formation by hybridoma cell) can continue under nongrowth conditions  Normally, most of mammalian cells cultures are mixed-growth associated (during growth phase until after growth ceases)

23. Products of Animal Cell Cultures

24.  Consists of high-molecular-weight proteins with or without glycosidic groups  There are enzymes, hormons, vaccines, immunobiologicals (monoclonal antibodies, regulators-lymphokines, virus vaccines), anticancer agents

25. Products Monoclonal Antibodies (Mab’s) Immuno- biological Regulators Virus vaccines Hormones  Produced by hybridoma cell  Used for diagnostic assay systems (determine drugs, toxins & vitamin); theraopeutic purposes & biological separations – chromatographic separations to purify protein molecules Interferon – anticancer glycoprotein (secreted animal cell or recombinant bacteria) Lymphokines Interleukines (anticancer agent) Prophylactics Virus is collected, inactivated and used as vaccine A weakened form will induce a protective response but no disease Large molecules: 50-200 amino acids Produce by hormone- synthesizing organ May also produce by chemical synthesis Example: Erythropoetin

26. Products EnzymesInsecticides Whole cells and tissue culture  Urokinase, rennin, asparaginase, collaginase, pepsin, trypsin, etc.. Produced some insect viruses that are highly specific and safe to envirionment Artificial organs and semi synthetic bone and dental structure