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192

exposed to any additional shear stress. Fraune et al.

(1991) have used a static PTFE hollow fiber membrane

module for the perfusion culture of various animal cell

lines including Spodoptera frugiperda but no further

details are given in their paper. An identical module

has been used by Klöppinger et al. (1990, 1991) for

the cultivation of Sf9 cells in a two stage process. A

maximum cell density of was achieved

in a silicone membrane-aerated 1.5-liter stirred tank

reactor for the production of wild-type Autographa

californica MNPV or recombinant A

different bioreactor system with membrane aeration via

microporous polypropylene tubings and an additional

hydrophilized polypropylene membrane for perfusion,

both mounted on a tumbling stirrer has been used for

the cultivation of IPLB-SF–21 AE cells. Cell densi-

ties of (Deutschmann & Jäger, 1991)

or (Deutschmann & Jäger, 1994) were

reached in a 1.2-liter stirred tank reactor with perfusion

rates of 3 and 4 reactor volumes per day, respective-

ly. Infection with recombinant Baculovirus and conse-

quent recombinant protein production was performed

at a cell density of up to (Jäger et al.,

1992) in bioreactors with 1.4 to 6 liter of working vol-

ume. Klöppinger et al. (1991) used a relatively high

multiplicity of infection (MOI) of 5 to 10, which pre-

supposes high titered virus stocks or large volumes of

virus suspension for infection. Jäger et al. (1992) used

an extremely low MOI of 0.015, which requires an

almost complete retention of virus within the bioreac-

tor in order to facilitate efficiently secondary infection

of all noninfected cells irrespective of the perfusion

rate. A retention of more than 99.9% was achieved

despite of the fact that the membrane pore size of

0.2 should theoretically allow a complete pen-

etration of the virus. More recently, an even lower

MOI of 0.001 has been used successfully for recom-

binant protein production with this perfusion system

(Jäger, unpublished results) (Figure 1). Schütz et al.

(1991) cultivated the Drosophila cell lines Kc and

Schneider–2, which have been used for stable transfec-

tion, in a 1.4-liter bioreactor of the same type result-

ing in maximum cell densities of and

respectively. Data of bioreactor cul-

tivations with internal membrane perfusion are sum-

marized in Table 1. The most significant drawback of

internal membrane perfusion is the limited potential

for scale-up. However, a scale-up to more than 200

liters of working volume appears to be feasible.

External membrane perfusion

In contrast to the special reactor configuration required

for internal membrane perfusion almost any type of

stirred tank or airlift bioreactor can be combined with

external membrane perfusion provided that the reac-

tor system has sufficient capacity to aerate higher

cell densities without increasing shear forces (intro-

duced by vigorous mixing or sparging) to a critical

extent. In addition, external membrane perfusion offers

the advantage that the microfiltration module can be

exchanged if the need arises from membrane blocking.

However, pumping of cells and circulation through

crossflow microfiltration systems is able to generate

substantial laminar shear stress to insect cells (Maiorel-

la et al., 1991). Therefore, choice of the crossflow sys-

tem and the circulation pump as well as adjustment of

flow rates are of particular importance.

First attempts to use external membrane modules

for medium exchange were based on a two stage sys-

tem for the production of wild-type virus (Klöppinger

et al., 1990). Propagation of Sf9 cells as well as virus

propagation were performed in silicone membrane-

aerated 1.5-liter stirred tank bioreactors. The reactor

used for virus propagation was perfused by means

of an internal microporous tubing system with 1

pore size, whereas the reactor for cell propagation was

equipped with an external Microgon MiniKros hollow

fiber crossflow filtration system. However, this system

was not used continuously but in a batchwise mode to

allow medium exchange prior to infection.

Continuous perfusion in a 4-liter Celligen reactor

combined with a home- designed tangential flow filtra-

tion system (de la Broise et al., 1991) resulted in den-

sities of (Massie et al., 1992).

However, the product yield, obtained when cells were

infected at was reduced to 50–75%

when compared to batch culture. This process was

further optimized by introduction of different spiral-

like tangential flow systems with or without integrated

membrane aeration (Caron et al., 1994). Perfusion at

rates between 1 and 4 reactor volumes per day was used

in the cell propagation phase as well as in the recombi-

nant protein production phase. 45 minutes after infec-

tion the tangential flow system was also used to con-

centrate cells before the initial culture volume was

reconstituted. Again, a reduced product yield of 75%

compared to spinner flasks was observed. In contrast,

Cavegn & Bernard (1992) obtained a normalized yield

varying from 0.5–3.5 compared to batch culture and a

significantly increased cell specific productivity (1.5–8

193

194

195

fold) in a 4-liter stirred tank reactor aerated by direct

pulse sparging and equipped with a Microgon Cellflo

hollow fiber crossflow microfiltration system for perfu-

sion (Cavegn et al., 1992). Densities of approximately

could be reached at perfusion

rates of up to 5 reactor volumes per day. Usually cells

were infected with a MOI of 5 at a density of

Using a 5-liter BioFlow III stirred tank reactor, aer-

ated by direct sparging and an A/G Technology hollow

fiber crossflow system Sf9 cells were grown to a den-

sity of at relatively low perfusion rates

of 0.4 to 0.6 reactor volumes per day (Reuveny et al.,

1994). The same crossflow system was also used for

high density cultures with medium replacement some-

times combined with fed-batch. The latter cultivation

method was suggested to be combined with a second

reactor for viral infection in order to obtain maximum

protein yields in minimum time.

Using a directly sparged 1.2-liter Cytoflow stirred

tank reactor combined with a hollow fiber crossflow

microfiltration system Sf9 and IPLB-SF–21 AE cells

were grown to maximum densities of

(Guillaume et al., 1992). Infections with a MOI of 1

were performed at The cell specific

productivity was comparable to batch cultures. The

same bioreactor system has also been used for the cul-

tivation of SPC-Mb 92 cells which were grown to a

maximum density of (Deramoudt et al.,

1994). Infected at a cell density of with

a MOI of 1 an 8.6 fold increase of the cell specific

productivity was observed.

IPLB-SF–21 AE were cultivated in a 10-liter air-

lift reactor equipped with a Prostak crossflow micro-

filtration system for medium perfusion (Hellenbroich,

1995). A maximum cell density of was

reached at a perfusion rate of 3 reactor volumes per

day. Cells were infected at a density of

with a MOI of 0.44. Due to the relatively small virus

196

inoculum cells continued to grow to

The yield of a recombinant interleukin–2 variant was

comparable to that obtained in experiments utilizing

membrane-aerated stirred tank reactors. In contrast,

Sf9 cells grown in a 23-liter airlift reactor with medi-

um perfusion showed a markedly reduced recombi-

nant protein production when compared to low density

batch cultures in the same reactor (Schlaeger et al.,

1992).

Perfusion processes using external membrane fil-

tration systems are summarized in Table 2. A wide

range of culture media with significantly differing

nutrient contents has been used for the cultivation of

insect cells with medium perfusion. Whereas IPLB-

SF-21 AE cells had to be perfused with 3 reactor vol-

umes per day to reach a cell density of

(Hellenbroich, 1995) the same cell density could be

achieved in batch culture by improved media formula-

tions (Ackermann et al., 1994). Due to this fact it is very

difficult to compare the different perfusion processes

and to extrapolate the potential of the reactor configu-

ration.

In general, external membrane perfusion has

proved to be a vital alternative to batch and fed-batch

culture. The most critical point appears to be a scale-

down of such bioreactor systems. In this case, the

circulation frequency of cells becomes too high or the

membrane channels become too small and thus gener-

ate intolerable shear stress.

Cell immobilization

Different approaches have been used to immobilize

insect cells for the production of virus and recombinant

proteins. One method is to encapsulate the cells by

alginate or other polymers (King et al., 1988, 1989).

This procedure is reviewed in more detail in a separate

chapter of this issue.

Another method is to grow cells by making use of

their potential to adhere to suitable carrier matrices.

Cells attached on these carriers can be cultivated in

various bioreactor systems depending on the properties

of the carriers with respect to size, shape and specif-

ic weight. Most frequently used are stationary (fixed,

packed) bed bioreactors and fluidized bed bioreactors.

Kompier et al. (1991a,b) cultivated IPLB-SF–21 AE

cells on non-woven fabric discs in a stationary bed reac-

tor for the production of recombinant

This small reactor consisted of a 50 ml column contain-

ing the stationary bed and a 350 ml membrane-aerated

medium reservoir. The medium was circulated with

a peristaltic pump. Even smaller columns of approxi-

mately 20 ml and filled with glass spheres were used

for the cultivation of BTI-Tn 5BI–4 cells (Shuler et

al., 1990). This column was connected to a small bub-

ble column. The culture medium was circulated by

means of a peristaltic pump or by the airlift itself.

More recently, this combination of two vessels was

replaced by introducing the bed directly into a split

flow airlift bioreactor of 210 ml volume (Chung et al.,

1993a). Although it is theoretically possible that all of

these reactor configurations can be run continuously

without complications, all systems were run in batch

mode at cell densities of or less. All

reactor systems contained solid particles without pores

as matrices which allow growth of cells only on the

surface. However, the question remains unanswered

whether insect cells show identical growth and pro-

ductivity at increased densities especially when limited

surface areas force cells to grow in multiple layers.

As an alternative, cells can be immobilized on

spherical macroporous carriers made of borosilicate

glass (SIRAN® , Schott) or hydrophobic polyethylene

(Cytoline–1® , Pharmacia) and cultivated in fluidized

bed bioreactors (Jäger, unpublished results). IPLB-SF–

21 AE and SPC-Bm36 were cultivated in a 0.25-liter-

fluidized-bed reactor described for the cultivation of

mammalian cells (Lüllau et al., 1994). Cell densities

of approx. were achieved based on the

total reactor volume or based on the

available volume inside of the macroporous carriers.

As shown in Figure 2 for the cultivation of IPLB-SF–

21 AE cells the growth rate as well as the produc-

tion rate of the recombinant protein are significantly

reduced when compared to perfused suspension culture

in stirred tank bioreactors (Jäger et al., 1992). It can

be assumed that in contrast to the surrounding culture

medium there might be insufficient oxygen concen-

trations inside the carriers with almost tissue-like cell

densities. The unsatisfactory results in the fluidized bed

could be a result of the fact that insect cell growth and

baculovirus-directed protein production are strongly

dependent on sufficient oxygen supply (Deutschmann

& Jäger, 1994; Jäger & Kobold, 1995).

Conclusion

High density perfusion culture of insect cells for the

production of recombinant proteins has proved to be an

attractive alternative to batch and fed-batch processes.

197

A comparison of the different production processes

is summarized in Table 3. Internal membrane per-

fusion has a limited scale-up potential but appears

to the method of choice in smaller lab-scale produc-

tion systems. External membrane perfusion results in

increased shear stress generated by pumping of cells

and passing through microfiltration modules at high

velocity. However, using optimized perfusion strate-

gies this shear stress can be minimized such that it is

tolerated by the cells. In these cases, perfusion cul-

ture has proven to be superior to batch production with

respect to product yields and cell specific productivity.

Although insect cells could be successfully cultivat-

ed by immobilization and perfusion in stationary bed

bioreactors, this method has not yet been used in con-

tinuous processes. In fluidized bed bioreactors with

continuous medium exchange cells showed reduced

growth and protein production rates.

For the cultivation of insect cells in batch and fed-

batch processes numerous efforts have been made to

optimize the culture medium in order to allow growth

and production at higher cell densities. These improved

media could be used in combination with a perfusion

process, thus allowing substantially increased cell den-

sities without raising the medium exchange rate. How-

ever, sufficient oxygen supply has to be guaranteed

during fermentation in order to ensure optimal produc-

tivity.

Acknowledgment

I am grateful to S. Grammatikos for critical reading of

the manuscript.

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vja@gbf-braunschweig.de

Cytotechnology 20: 199–208, 1996. 199

Immobilization of insect cells

Jian-Yong Wu

1,3

& Mattheus F. A. Goosen

1,2

Department of Chemical Engineering, Queen’s University, Kingston, Ontario, K7L 3N6, Canada;

Sultan

Qaboos University, College of Agriculture, P.O. Box 34, Al-Khod 123, Muscat, Sultanate of Oman;

Current

Address: Department of Applied Biology & Chemical Technology, Hong Kong Polytechnic, Kowloon, Hong Kong

Key words: immobilized insect cell culture

Introduction

The production of baculoviruses and recombinant pro-

teins in insect cell culture has been mostly carried out

in culture systems with freely-suspended cells. Sus-

pended culture systems usually suffer from a number

of disadvantages, such as the fluid mechanical cell

damage from agitation and air-sparging, low cell and

product concentrations, and low productivity. Another

limitation is that the method is only suitable for the

growth of non anchorage-dependent cell (e.g., Sf–9

cells). These drawbacks can be eliminated with the

exploitation of cell immobilization techniques, such

as the attachment of cells on solid surfaces, entrap-

ment of cells in polymer gels, and encapsulation of

cells in artificial membranes. With immobilized cells,

a 10-fold or more increase in the cell and produc-

tion concentrations can be achieved (e.g., Dean et al

1987; Reiter et al

1991; and Park & Stephanopoulos,

1992). The technology will also allow for simpler cell-

medium separation and product purification processes.

In addition, immobilization of cells in porous supports

and microencapsules could also prevent the cell from

mechanical damage due to mechanical agitation and

gas sparging.

There have been extensive studies on immobiliza-

tion of animal cells and its application in large-scale

production of mammalian cells and associated prod-

ucts, such as hybridoma cells in the production of

monoclonal antibodies (Reuveny, 1985; Heath and

Belfort, 1987). The development of immobilized insect

* To whom correspondence should be addressed.

cell culture, however, is still in its infant stage. This

is not surprising since it is only recently that insect

cell culture has been recognized as an important tool

in the biotechnology industry (Luckow and Summers,

1988). Other factors responsible for the fewer studies

on insect cell immobilization may be related to the type

of insect cells used for large-scale production and the

mechanism of virus and recombinant protein produc-

tion. The cell lines used predominantly in baculovirus

and recombinant protein production, i.e., those derived

from Spodoptera frugiperda insect, Sf–21 and Sf–9,

grow well in suspension, which undermines the advan-

tages of immobilized culture. On the other hand, pro-

duction of cell products involves viral infection and

subsequent lysis of the cell. Immobilized cell culture

can thus only be used for one batch operation. With

the development of anchorage-dependent insect cells

for virus and protein production, and processes of pro-

ducing secreted cell products without cell lysis, immo-

bilization technology will become more attractive for

large-scale production.

In this chapter, we will primarily focus on tech-

niques for animal cell immobilization in particulate

supports, including microcarrier beads and microcap-

sules, with emphasis on their applications in insect cell

culture. Typical immobilization materials and meth-

ods, immobilized cell bioreactors and the major con-

cerns associated with each technique will be addressed.

The review will conclude with an outline of the studies

conducted in our laboratory on insect cell immobiliza-

tion techniques.

200

Cell immobilization systems

A variety of systems can be employed for cell and

enzyme immobilization. These include, for example,

microcarriers (van Wezel, 1967), gel entrapment (Guo

et al., 1990), hollow fibers (Knazek et al., 1972), and

encapsulation (Lim and Moss, 1981). In principle,

these methods fall into one of two categories: entrap-

ment in a three dimensional polymer matrix or cap-

ture behind a semipermeable membrane. Among these

methods, microcarriers and gel entrapment are proba-

bly the most popular. However, one common problem

with both is the leakage of the immobilized biocata-

lyst. Hollow fibers and microencapsulation overcome

this problem by separating the cells from converted

medium with a semipermeable membrane. Our atten-

tion in this section will be on the immobilization of

animal cells in particulate carriers, including micro-

carriers, porous beads and microcapsules (Table 1).

Microcarriers

The introduction of microcarriers to animal cell cul-

ture is a major breakthrough in the development of

anchorage-dependent animal and mammalian cell cul-

ture processes. The traditional method for large-scale

production of anchorage-dependent animal cells was

to grow the cells on the inside surfaces of rotating bot-

tles, or roller bottles. Major disadvantages of the roller

bottle system include a very low surface-to-volume

ratio, variable quality of the cell products and exten-

sive labor costs required for processing large numbers

of bottles: In a microcarrier culture, on the other hand,

cells are attached on the surface of small carrier beads

suspended in the medium. The small carrier beads,

with a diameter of a few hundred microns, provide

a large surface area for cell attachment. For instance,

the surface-to-volume ratio of a microcarrier culture

is about at 5 g/l particle concentration, com-

pared to 1.2 cm

–1

for a roller bottle (Butler, 1987).

Apart from a significant increase in the surface-to-

volume ratio, use of microcarriers has made it possible

to cultivate anchorage-dependent cells in a homoge-

neously suspended condition, which greatly facilitates

large-scale production.

Microcarrier culture technology was first report-

ed by van Wezel (1967), nearly three decades ago.

He cultivated mammalian cells adhering on DEAE-

Sephadex

A–50 beads in suspension culture. The

microcarrier chosen by van Wezel (1967), i.e., DEAE-

Sephadex A–50, was designed primarily for ion

exchange chromatography. These microcarrier beads

consist of a dextran matrix with a net positive surface

charge which is considered necessary for cell attach-

ment. The microcarriers were found suitable for cell

growth at particle concentrations no greater than 1 g/l.

Exceeding that level of beads loading was found to

cause inhibitory effects on cell growth. This adverse

effect was believed to be related to the charge density

on the bead surface. By reducing the surface charge

of the microcarriers, Levine et al. (1977) were able

to eliminate this toxic effect, achieving cell attach-

ment and growth at particle concentrations up to 5 g/l.

This breakthrough made it feasible to grow anchorage-

dependent cells to high cell densities to suspension

culture bioreactors.

Polymeric products, such as dextran (polysaccha-

ride), cellulose, Gelatin (polysaccharide) and plastic

(polystyrene) have been primary matrix materials for

cell immobilization (Table 2). Particularly, the dextran-

based microcarriers, Cytodex 1, 2, 3 (Pharmacia Fine

Chemicals, Uppsala, Sweden), have been most widely

used in large-scale cell culture as well as laboratory

research. The most important physical and chemical

properties of microcarriers particles may include sur-

face charge, density and size. Surface charge, as men-

tioned earlier, is crucial for cell attachment and cell

growth on the microcarriers. The optimal charge den-

sity is 2.0 meg/g (Butler, 1987). Usually the density

of the microcarrier beads is relatively low (typical-

ly 1.03 g/l, slightly higher than the liquid medium)

to facilitate suspension with gentle agitation. Heavier

materials requiring high stirring speeds to suspend may

cause cell damage. The diameter of the microcarriers

should conform to a high surface-to-volume ratio, there

should be sufficient surface area on each microcarri-

er for a single cell to divide for several generations,

the manufacture process should also be inexpensive.

Diameters in the range of 150–220 have been found

to be desirable. Other properties, such as microcarri-

er transparency, size distribution and rigidity are also

important concerns for cell attachment, growth in sus-

pension, visual observation, and cell growth and viabil-

ity measurement. In addition, non-toxicity is essential

for cell survival as well as for medical applications of

cell products.

A major drawback with current microcarriers is that

they cannot be reused (i.e., cleaning and sterilization).

This results in considerable increase in the cost of the

culture process (e.g., $50/l with 5 g/l beads loading,

Glacken et al., 1983). In addition, one of the major

concerns with microcarrier culture is mechanical dam-

201

age of the cell attached on the carrier surface due to

fluid shear stresses and possibly collision between the

carrier beads (Croughan et al., 1988).

The kinetics of animal cell growth on microcarriers

was found to be essentially the same as in monolayer

culture, but much higher cell densities can be achieved

if the medium is replenished (e.g., medium perfusion)

(van Wezel, 1972). The choice of a suitable micro-

carrier depends on the type of cells (e.g., plating effi-

ciency and morphology) and the purpose of the culture

(product harvest or cell biology studies). This will in

turn affect the choice of the culture vessels. Although

in principle microcarrier culture can be contained in

virtually any type of cell culture vessel (static mono-

layer or suspension), its advantages are best realized

in stirred suspension culture. To avoid cell detachment

and fluid mechanical damage, the stirring action should

be gentle, and evenly distributed, with minimum agi-

tation.

Porous carriers

Although microcarrier technology has many advan-

tages for animal cell culture, it still has some draw-

backs. First of all, the technique is only suitable for

anchorage-dependent cells. Secondly, cells attached