the cell culture environment
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The cell culture environment
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Factors affecting cell behaviour in vivo
The local micro-environment
Cell-cell interactions
Tissue architecture Tissue matrix
Tissue metabolites
Locally released growth factor and hormones
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(2) Culture Surface
Most adherent cells require attachment to
proliferate
Change charge of the surface
Poly-L-lysine
Coating with matrix proteins
Collagen, laminin, gelatin, fibronectin
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(3) Media formulation
Initial studies used body fluids
Plasma, lymph, serum, tissue extracts
Early basal media
Salts, amino acids, sugars, vitamins supplemented
with serum
More defined media
Cell specific extremely complex
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DMEM
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Media Formulation
Inorganic ions Osmotic balancecell volume
Trace Elements Co-factors for biochemical pathways (Zn, Cu)
Amino Acids Protein synthesis
Glutamine required at high concentrations
Vitamins Metabolic co-enzymes for cell replication
Energy sources glucose
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Serum provides the following
Basic nutrients
Hormones and growth factors
Attachment and spreading factors Binding proteins (albumin, transferring)
carrying hormones, vitamins, minerals, lipids
Protease inhibitors pH buffer
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Freshney.(1992) Animal Cell Culture.
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(4) The gas phase
Oxygen
Aerobic metabolism
Atmospheric 20%
Tissue levels between 1-7%
Carbon dioxide
Buffering
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(5) pH Control
Physiological pH 7
pH can affect
Cell metabolism
Growth rate
Protein synthesis
Availability of nutrients
CO2acts as a buffering agent in combinationwith sodium bicarbonate in the media
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Mouse Myogenic Cells (H-2Kb)- grown on fibronectinin 8 well slide in culture, fixed and stained for desmin
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Contamination
Minimise the risk
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Sources of Contamination
Bacteria
Fungi
Mould
Yeast Mycoplasma
Other cell types
Free organisms, dust particles or aerosols
Surfaces or equipment
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Class II
Biological
Safety
Cabinet
Protection of
personnelenvironment
product
Class 1 Cabinets
protect the
product only
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Vertical
Laminar AirflowAir Barrier
Exhaust Fan
Exhaust
HEPA Filter
Laminar Flow
Fan
Laminar
HEPA Filter
Class II
Biological
Safety
Cabinet
HEPA filters
Laminar flow
NATA certified
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Sitting or standing with no movement, wearingcleanroom garments, an individual will shedapproximately 100,000 particles of 0.3um and largerper minute. The same person with only simple arm
movement will emit 500,000 particles. Average armand body movements with some slight legmovement will produce over 1,000,000 particles perminute; average walking pace 7,500,000 particles perminute; and walking fast 10,000,000 particles per
minutes. Boisterous activity can result in the releaseof as many as 15x106to 30x106particles per minuteinto the cleanroom environment.
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Aseptic Technique 1
Controlled environment
Traffic, air flow
Sterile media and reagents
Avoids aerial contamination of solutions
Avoids manual contamination of equipment
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Aseptic Technique 2
Minimise traffic
Clear work area
70% ethanol swab
Minimise work area (field of vision)
Keep work area clean
Do not lean over open vessels
UV irradiation before and after
Only use disposable equipment once
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Aseptic Technique 3
Minimise exposure to air
Flame bottles if on open bench
Avoid repeated opening of bottles
Avoid liquid accumulation around necks and lips ofbottles
Avoid excessive agitation
Only one cell type at a time
Do not open contaminated solutions
No burner in hood