1. Modes of culture for high cell densities Chapter 10 ‘The Basics’

2. 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

3. Heterogeneous system

4. Detrimental environment for cell growth -Depletion of an essential nutrient -Accumulation of an inhibitor -Complete coverage of available growth surface

5. 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

6. What is a Continuous Culture? Open system – continuous feed of medium and removal of ‘spent’ medium Cell growth - longer period in CC>BC

7. Types of Continuous Culture Chemostat culture – Cells and spent medium are continuously removed - State of culture is dependent upon flow rate of fresh medium

8. 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

9. 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

10. What are Microcarriers? Microcarriers are microscopic particles (diameter = 200 μm) Maintained in suspension in liquid medium Dextran, Collagen and Plastic

11. 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

12. 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

13. Extraction of cells from Microcarriers Detachment by either trypsin or collagenase treatment Detached cells separated by sieving through a nylon mesh

14. Immobilization of nonanchorage-dependent cells Protection against mechanical stress Ease of continuous operation Isolation of products

15. 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

16. 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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