Scaling up animal cell culture Chapter 9 from ‘The Basics’ = Chapter 26 from ‘Culture of Animal Cell Culture’ Why scale-up cultures? What are small cultures? Experiments requiring large number of replicates Determination of concentration-dependent effects Multi-well plates accommodate culture volumes 2-3mls What are small cultures? Experiments require multiple samples - To observe cell growth and to perform substrate or product assays - Culture volumes – 100 ml - Conducted in T-flasks or spinner flasks in Incubator Why scale-up cultures? Required to produce substantial quantities of a cell product – such as virus or glycoprotein or enzymes Two approaches - A Multiple process involving 1000 culture flasks (100 ml) - A Unit process involving 100-liter fermenter Factors to control in Fermenters/Bioreactors? Commercial Cultures 1 – 5 liters Oxygen supply, temperature control, pH control and culture mixing Electrodes Choice of Bioreactor Equipment designed to grow cells in culture Type and design of bioreactor and mode of operation - Depends on cell densities and productivity The Stirred tank reactor (STR) Simplest and most widely used Consists of cylindrical vessel with a stirrer (a pot and paddle) Designed differently for animal cells and for bacterial or fungal cultures Bioreactors The Stirred tank reactor (STR) Large-scale animal cell culture processes – 10,000 liters - Stainless steel Bench-top STRs (1-5 liters) - Glass What are the parameters that control adequate culture growth? Agitation – Bubble bursting on culture surface resulting from culture aeration Stirring speed is low – rotation of a suspended bar by a magnetic stirrer - are not suitable in larger volumes Impellers – vertical and horizontal movement Parameter - Agitation Maximum stirring rates for suspension – 100- 150 rpm Microcarrier - < 40 rpm (suspension and anchorage dependent cells) Round bottom (animal cells) and flat bottomed vessels (bacterial cells) Parameter – Temperature control Thermocirculator – pumps heated water around an outer jacket - Larger fermenters – pumps water through coiled pipes within culture Circulating warm air Low volume fermenters – External heating pads Parameter – pH control Optimal pH - 7.4 – for maximum growth Enriched Co2 atmosphere decreases pH fluctuations 1-litre culture @ 2x106 cells/ml @ gas flow of 100 ml/min Parameter – pH control Direct acid or alkali addition Net acidic production (lactic acid) from cellular metabolism – alkali (NaHCO3) HCl is added Computer-controlled pump or gas valve to a pre-set pH value Rotameters indicate rate of gas flow – controlled by flow regulators (fig-9.6) Parameter – Oxygen supply Major problem Gas diffusion from head space through culture surface decreases Oxygen transfer rate (OTR) across liquid surface > Oxygen utilization rate (OUR) What is Sparging? Common in bacterial cultures Cell damage is caused – bubble bursting Leads to foaming Alternative method – Surround gas sparger by fine-mesh cage Parameter – Oxygen supply Control of oxygen supply by a sterilizable oxygen probe Solubility of oxygen in media can be increased by addition of some liquid perfluorocarbons Indirect aeration – Oxygen supply Indirect aeration involves medium sparging Suitable for media recirculation Oxygen supplied by gas diffusion through thin walled silicone tubing 1 meter of tubing/2 liters of culture (adequate twining or binding) Alternative types of Bioreactors Airlift fermenter Consists of tall column with an inner draught tube Fluid circulation is provided by stream of air Less bubbling or foaming Production of monoclonal antibodies Alternative types of Bioreactors Hollow-fiber Cartridge made up of several thousand minute capillary-like plastic tubules (fibers) with perfusable membrane walls Anchorage-dependent and independent cells Alternative types of Bioreactors Packed-bed or fixed-bed bioreactor Support matrix for attachment and growth of anchorage dependent cells Continuous flow of medium Glass bead column, ceramic and fluidized- bed bioreactors Alternative types of Bioreactors Glass bead column Glass column-glass beads with a diameter of 3-5 mm Medium is recirculated through packed bed by pump and oxygenated by air Alternative types of Bioreactors Ceramic bioreactor Series of channels run through ceramic cylinder Each channel is a square with 1mm sides and an inner surface area for cell attachment No longer in use Alternative types of Bioreactors Cell Cube Stack of 20 cm2 polystrene plates spaced 1mm apart by rigid spacers Cells attach to either side of plate Flow of culture medium between plates Production of vaccines Alternative types of Bioreactors Fluidized-bed reactor Upward flow of medium recirculated by pumping Cells immobilized or entrapped in beads are held in suspension in column This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). 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