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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