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Large Scale Cultivation of Animal Cells

Many animal cell lines can be cultivated in suspension culture similar to microorganisms. Some cell types (e.g. typical normal diploid cells) are anchorage- dependent (i.e. they require a surface to grow on).

Such cells are grown on the inside surface of plastic or glass bottles or on the surface of micro carriers suspended in liquid medium. An artificial culture environment maintains its optimum pH (6.7 to 7.9), dissolved O2 (20-80%) and temperature. The animal cells are sensitive to shear forces.

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Shear occurs due to stirring and sparging. Cells attached to air bubbles are exposed to enormous forces when bubbles leave the bulk liquid at the surface and burst due to decompression.

Cells in spared cultures can be protected from shear forces by using medium with a high viscosity. This can be achieved through high cell density (>107 cells/ml).

Similar to microorganisms, the animal cells can also be cultivated as batch, fed-batch and continuous culture. Continuous process can be carried out as a chemostat or perfusion culture (i.e. a continuous culture when biomass is retained in a reactor and cell- free culture liquid is removed).

Methods for Scale-up of Cell Culture Process :

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In batch culture there are various methods for scaling up of animal cell culture process. These methods include roller bottles with micro carrier beads (for adherent cell culture) and spinner flasks (for suspension culture).

(a) Roller Bottles:

Early commercial production with anchorage-depen­dent cells was often performed in roller bottles. Inside the roller bottles animal cells adhere to curved surface area of microcarrier beads. Consequently, this increases the total surface area for available spaces for the growth of the cells. Typically, a surface area of 750-1500 cm2 with 200-500 ml medium will yield 1-2 x 108 cells. Moreover, a large surface area could be obtained by the use of microcarriers in stirred tank reactor.

The roller bottles are well attached inside specialised CO2 incubators. The attachments rotate the bottles along the long axis. The entire cell monolayer is exposed to the medium after each full rotation of the bottle. The volume of the medium superficially covering the monolayer is sufficient for cell growth.

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Microcarrier Beads:

Microcarriers are also called microcarrier beads because these are small spherical particles (having 100-200 micrometer diameter) looking like beads. For animal cell culture microcarrier beads are used to increase the space for cell growth.

This results in an increase in number of the adherent cells per flask. These beads are made up of dextran or glass. These beads can also be used with spinner culture flasks as they are buoyant.

At the recommended concentration when microcarriers are resuspended, they provide 0.24 m2 area for every 100 ml of culture flask

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This condition supports the growth of adherent cells to a very high density. Otherwise they shall be crowded and cause problem. The rapid cell growth quickly exhausts growth medium.

(b) Spinner Cultures:

Scaling-up of process requires several intermediate steps. The first step is the transfer of cells from stationary culture to shake flask or spinner flask. The spinner flask was originally developed to provide gentle stirring of microcarriers. But now it is used for suspension culture also.

The spinner flask consists of a flat surface glass flask. A teflon paddle (impeller) hangs down in the flask from the lid and remains suspended without touching the bottom. The impeller rotates and agitates the medium when flask is put in a magnetic stirrer.

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The spinner flask used at commercial scale consists of one or more side arms for taking out samples and decantation as well. The cells are

Alternatives to Spinner Culture :

1. A single unit process in a well-adapted bioreactor is required.

2. A compact-loop bioreactor with marine impeller would satisfy these conditions.

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