Modes of culture for high cell densities
Chapter 10 ‘The Basics’
What is a Batch Culture?
Cells are inoculated – culture left for several days – until final density is reached
Nothing is added or removed during culture
Substrates get used up and products are secreted from cells
Heterogeneous system
Detrimental environment for cell growth
- Depletion of an essential nutrient
- Accumulation of an inhibitor
- Complete coverage of available growth surface
What is Fed-batch culture?
Cell growth – nutrient supply or removal of by-products
Cell yield will reach high densities
In open system/Fed-batch culture – involves controlled nutrient feeding
Partial media changes at regular intervals
What is a Continuous Culture?
Open system – continuous feed of medium and removal of ‘spent’ medium
Cell growth - longer period in CC>BC
Types of Continuous Culture
Chemostat culture – Cells and spent medium are continuously removed
- State of culture is dependent upon flow rate of fresh medium
Types of Continuous Culture
Perfusion culture – Cells are retained in fermenter
- Medium is pumped continuously
- Cells are retained
- Becoming popular for large-scale production
- Attains high cell density
- Cell separator
Cell immobilization
Immobilization of cells on or inside particles
Allows attachment of cells to solid surface
Anchorage dependent cells
Entrapment of cells in small beads
What are Microcarriers?
Microcarriers are microscopic particles (diameter = 200 μm)
Maintained in suspension in liquid medium
Dextran, Collagen and Plastic
Characteristics of Microcarriers
Small – to maximize the available surface area for cell growth
Light – to allow easy suspension in culture medium
Transparent – to allow easy observation of cell attachment and growth
Charged – to allow cell attachment onto surface
Porous microcarriers
Dextran microcarriers (example: Cytodex) are microporous
- Pore size is not sufficient to allow cells to colonize the interior of beads
Gelatin microcarriers (example: Cultispher) are macroporous
- Increased surface area for attachment - Interior environment to protect cells against
adverse shear forces
Extraction of cells from Microcarriers
Detachment by either trypsin or collagenase treatment
Detached cells separated by sieving through a nylon mesh
Immobilization of nonanchorage-dependent cells
Protection against mechanical stress
Ease of continuous operation
Isolation of products
Immobilization of nonanchorage-dependent cells
Cell entrapment –
- Mixing and agitating suspension of cells in warm agarose with hydrophobic liquid – paraffin oil
- Forms solid beads of agarose (100-200 μm) containing suspended cells
- Secreted cellular products – separated from entrapped cells
Immobilization of nonanchorage-dependent cells - Encapsulation
Cells – enclosed in semipermeable membrane
Cells + sodium alginate drip into calcium alginate
Polylysine creates an outer semipermeable membrane
Monoclonal antibodies accumulate inside the bead matrix – can be extracted easily
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