42 Large-Scale Cell Culture
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from 2 x 10 5 to 6 X 10 6 cells/ml during 7 days culture at 2rc. With progression of culture, some growth inhibition was observed, and this limitation coincided with nutrient depletion, in particular glucose and
glutamine, and the generation of toxic substances.
Subprotocol 5
Internal Membrane Perfusion Culture
In this system, continuous medium exchange by internal membrane perfusion does not require additional pumping of cells through a filtration
system.
Materials
- Perfusion module (hollow fiber membrane, silicone membrane, Equipment
microporous polypropylene tubing, and hydrophilized polypropylene
membrane are some of the perfusion modules used)
- Stirred tank bioreactor
- Culture media
Solutions
Procedure
1. Harvest cells from a stock culture.
2. Place the cells in a culture system.
3. Run the culture system.
Results
With this culture system, a cell density of more than 10 7 cells/ml will be
achieved. However, this system has a limited potential for being scaled
up.
Comments
Perfusion culture is useful for keeping cells free from toxic metabolic
products, which may accumulate in the culture medium. However, in insect cell culture, there appears to be no critical accumulation of cytotoxic metabolites, such as ammonium. Nevertheless, the use of perfusion
culture in insect cell culture has some advantages, especially if the culture is used for the investigation of recombinant proteins by means of a
baculovirus vector system. In this case, perfusion culture reduces the
residence time of secreted recombinant proteins in the bioreactor, and
minimizes product degradation. In addition, it simplifies the first step of
downstream processing of the products, namely the separation of a secreted recombinant protein from cell debris. Furthermore, because of
393
from 2 x 10 5 to 6 X 10 6 cells/ml during 7 days culture at 2rc. With progression of culture, some growth inhibition was observed, and this limitation coincided with nutrient depletion, in particular glucose and
glutamine, and the generation of toxic substances.
Subprotocol 5
Internal Membrane Perfusion Culture
In this system, continuous medium exchange by internal membrane perfusion does not require additional pumping of cells through a filtration
system.
Materials
- Perfusion module (hollow fiber membrane, silicone membrane, Equipment
microporous polypropylene tubing, and hydrophilized polypropylene
membrane are some of the perfusion modules used)
- Stirred tank bioreactor
- Culture media
Solutions
Procedure
1. Harvest cells from a stock culture.
2. Place the cells in a culture system.
3. Run the culture system.
Results
With this culture system, a cell density of more than 10 7 cells/ml will be
achieved. However, this system has a limited potential for being scaled
up.
Comments
Perfusion culture is useful for keeping cells free from toxic metabolic
products, which may accumulate in the culture medium. However, in insect cell culture, there appears to be no critical accumulation of cytotoxic metabolites, such as ammonium. Nevertheless, the use of perfusion
culture in insect cell culture has some advantages, especially if the culture is used for the investigation of recombinant proteins by means of a
baculovirus vector system. In this case, perfusion culture reduces the
residence time of secreted recombinant proteins in the bioreactor, and
minimizes product degradation. In addition, it simplifies the first step of
downstream processing of the products, namely the separation of a secreted recombinant protein from cell debris. Furthermore, because of