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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