The Cell Culture Environment

8/10/2019 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

The Cell Culture Environment

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