Considerations in Using Animal Cell Culture

•Structure and biochemistry of animal cells

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.

Figure 1: structure of animal cell

Structure and function of organellesOrganelles Function

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.

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.

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.

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)

Methods Used for The Cultivation of Animal Cells

Methods Used for The Cultivation of Animal Cells

Differ significantly from those used with bacteria, yeast and fungiTissue 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 aggregateGrow 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.

Steps in animal cells cultureSteps in animal cells culture

1)Excised tissues are cut into small pieces (~ 2mm3)

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 centrifuged5)Cells are re-suspended in growth medium 6)Placed in T-flasks or roller bottles

Anchorage-dependent cells : Cells growing on support surfacesNonanchorage-dependent cells : Cells grow in suspension culturePrimary culture: The cells that directly derived from excised tissuesSecondary culture: A cell line obtained from the primary culture

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

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

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

Hybridoma CellObtained by fusing lymphocytes (normal blood cells that make antibodies) with myeloma (cancer) cellsLymphocytes producing antibodies grow slowly and are mortalAfter fusion with myeloma cells, hybridomas become immortal, can reproduce and produce antibodies.

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

SerumA 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.

Serum’s function:

1.To stimulate cell growth and other cell activities by hormone and growth factors2.To enhance cell attachment by certain proteins such as collagen and fibronectin3.To provide transport proteins carrying hormones, minerals and lipids

Kinetic Growth of Mammalian Cell Culture

No. Cells Growth condition1. Mammalian cells 37°C, pH ~7.3

Doubling time: 12 – 20 hNeed to be gently aerated and agitatedBuffer used: Carbonate buffer/CO2-enriched air/HEPES

2. Insect cells 28°C, pH 6.23. Fish cells 25°C-35°C, pH 7 – 7.5

Similar to microbial growthStationary phase is relatively shortConsentration 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 daysProduct formation (monoclonal antibody formation by hybridoma cell) can continue under nongrowth conditionsNormally, most of mammalian cells cultures are mixed-growth associated (during growth phase until after growth ceases)

Products of Animal Cell Cultures

Products of Animal Cell Cultures

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

Produced by hybridoma cellUsed 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)

ProphylacticsVirus is collected, inactivated and used as vaccineA weakened form will induce a protective response but no disease

Large molecules: 50-200 amino acidsProduce by hormone-synthesizing organMay also produce by chemical synthesisExample: Erythropoetin

Urokinase, rennin, asparaginase, collaginase, pepsin, trypsin, etc..

Produced some insect viruses that are highly specific and safe to envirionmentArtificial organs and semi

synthetic bone and dental structure