Chapter 42
Large-Scale Cell Culture
The large-scale culture of cells requires the optimization of various factors, such as cell density, cell viability, growth stage, cell nutrition, composition of media, dissolved oxygen concentration (DO), temperature,
pH, and osmotic pressure of the media. In addition, the cost of the media
is important for industrialization, and in this context, whether the media
can be sterilized by heating is important.
Of these factors, the DO level is important in the large-scale culture
of cells. According to Kamen et aI. (1991), insect cells require up to
1.5 mmollllh oxygen, and Jem et aI. (1997) have reported that a 50% DO
was appropriate for the production of insect cells and baculovirus polyhedral inclusion body (PIB) in a 5-1 bioreactor culture. However, the solubility of oxygen in culture media is poor. As a result, it becomes necessary to supply oxygen to the culture. A ring sparger is commonly used to
liberate small bubbles of oxygen into the culture medium. However, these
oxygen bubbles generate shear force when they burst. It has been determine that an antifoaming agent, such as Pluronic F-68 (Sigma), or methylcellulose have some effects in protecting cells against shear damage.
Insect cells are attracted to the surface of rising bubbles (Bavarian et aI.,
1991), and Pluronic F-68 is said to prevent the attachment of cells to the
bubbles.
The optimum levels of the various factors that need to be taken into
consideration are different for different cell lines. Therefore, it is necessary to determine the optimum conditions before scaling-up the culture.
For the production of viruses by insect cells cultured on a large scale,
a batch culture of cells is common. In this case, a fed batch culture is
often effective in obtaining a high cell density. Effective nutrients for the
feeding of cells are glucose and glutamine (Ackermann et aI., 1994),
yeastolate ultrafiltrate and an amino acid mixture (Bedard et aI., 1994).
There is no specific procedure for the large-scale culture of insect
cells. In any type of culture, the procedure consists of the preparation of
cells for seeding, and inoculation of cells. Inoculation is commonly done
so as to make the initial cell density of a large-scale culture 1-5 x 10 5 Iml
or more. There is no specific method of inoculating cells and maintaining cell culture of a large scale. For the preparation and operation of
different culture systems, refer to the manuals provided by the manufacturers of the culture systems.
PROTOCOL
Large-Scale Cell Culture
The large-scale culture of cells requires the optimization of various factors, such as cell density, cell viability, growth stage, cell nutrition, composition of media, dissolved oxygen concentration (DO), temperature,
pH, and osmotic pressure of the media. In addition, the cost of the media
is important for industrialization, and in this context, whether the media
can be sterilized by heating is important.
Of these factors, the DO level is important in the large-scale culture
of cells. According to Kamen et aI. (1991), insect cells require up to
1.5 mmollllh oxygen, and Jem et aI. (1997) have reported that a 50% DO
was appropriate for the production of insect cells and baculovirus polyhedral inclusion body (PIB) in a 5-1 bioreactor culture. However, the solubility of oxygen in culture media is poor. As a result, it becomes necessary to supply oxygen to the culture. A ring sparger is commonly used to
liberate small bubbles of oxygen into the culture medium. However, these
oxygen bubbles generate shear force when they burst. It has been determine that an antifoaming agent, such as Pluronic F-68 (Sigma), or methylcellulose have some effects in protecting cells against shear damage.
Insect cells are attracted to the surface of rising bubbles (Bavarian et aI.,
1991), and Pluronic F-68 is said to prevent the attachment of cells to the
bubbles.
The optimum levels of the various factors that need to be taken into
consideration are different for different cell lines. Therefore, it is necessary to determine the optimum conditions before scaling-up the culture.
For the production of viruses by insect cells cultured on a large scale,
a batch culture of cells is common. In this case, a fed batch culture is
often effective in obtaining a high cell density. Effective nutrients for the
feeding of cells are glucose and glutamine (Ackermann et aI., 1994),
yeastolate ultrafiltrate and an amino acid mixture (Bedard et aI., 1994).
There is no specific procedure for the large-scale culture of insect
cells. In any type of culture, the procedure consists of the preparation of
cells for seeding, and inoculation of cells. Inoculation is commonly done
so as to make the initial cell density of a large-scale culture 1-5 x 10 5 Iml
or more. There is no specific method of inoculating cells and maintaining cell culture of a large scale. For the preparation and operation of
different culture systems, refer to the manuals provided by the manufacturers of the culture systems.
PROTOCOL