culture environment
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The Culture Environment
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BSL4
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Components of the
Culture Environment
The Gas Phase
The Substrate
Growth surface The Medium / Media
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The Gas Phase
Short-term cultures
95% O2: 5% CO2
Long-term cultures
Air + CO2to 5%
Add sodium bicarbonate
Buffer in media
Cellular requirement for bicarbonate
Balanced salt solutions
Air - Hanks balanced salts: 0.35 g / l
5% CO2- Earless balanced salts: 2.2 g / l
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The Substrate
Glass Plastic (usually polystyrene)
Pretreated to make a hydrophilic surface
Net + charge
Many types
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Culture VesselsDescriptions and Surface Areas
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Vented Dish
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Multiwell Plates
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T-Flasks
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T-Flask Permeable Caps
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Chamber Slides
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Roller Bottles
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Roller Bottle Cross-Section
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Nunclon Cell Factory
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Filling the Nunclon Cell Factory
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The Substrate Glass
Plastic (usually polystyrene)
Pretreated to make a hydrophilic surface
Net + charge
Many types
Microcarriers
100 m diameter
Increase cell density / unit volume of media
Useful for bioreactors
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Microcarrier Beads with Attached Cells
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Stirrer Flasks for Microcarrier Beads
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Stirrer FlaskCross-Section
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The Substrate (cont.)
Feeder layers
Monolayer of fibroblasts, UV irradiated
Seed cells of interest on top
Porous culture inserts
Induce apical / basolateral polarity
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Porous Culture Inserts
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Porous Culture InsertsCross-Section
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The Substrate (cont.)
Feeder layers
Monolayer of fibroblasts, UV irradiated
Seed cells of interest on top
Porous culture inserts
Induce apical / basolateral polarity
Hollow fiber systems
Mimic tissue capillary bed
Histotypic culture
A way of generating 3D structure
Scale-up for bioreactors
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Hollow Fiber System
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CellMax Hollow Fiber System
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New Brunswick Scientific
Model BioFlo 110, 1.3 - 14 l
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New Brunswick Scientific
Model Bioflo 6000, 50 - 130 l
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Screen Capture of New Brunswick
Scientific BioControl System
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Jagadeesh Chandra, A.P., Sudhaker Samuel, R.D. 2009. Modeling,
Simulation and Control of Bioreactors Process Parameters
Remote Experimentation Approach. International Journal of
Computer Applications . 10: (0975 8887).
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The Substrate (cont.) Specialty proteins / Extracellular matrix proteins
Precoated onto plastic vessels
Attachment
Induction / maintenance of differentiated functions
Collagen (most commonly used) Coating on plastic
Sandwich
Gel
Fibronectin
Laminin
Polylysine
Matrigel (Becton-Dickinson)
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Attachment Problems - Spotting
Due to Air Bubbles
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Attachment Problems
Meniscus Rings
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Attachment Problems - Peeling
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Attachment Problems - Temperature Effects
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Attachment Problems - Vibration Effects
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Attachment Effects - Tilted Vessel Effect
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Hemocytometer Diagram
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Section of a Hemocytometer