Journal of Biotechnology 59 (1997) 39 52

Minireview

Plant cell suspension cultures: some engineering considerations

P.M. Kieran a,*, P.F. MacLoughlin b, D.M. Malone b
a

Biochemical Engineering Research Group, School of Biological Sciences, Dublin City Uni6ersity, Dublin 9, Ireland
b
Department of Chemical Engineering, Uni6ersity College Dublin, Belfield, Dublin 4, Ireland
Received 1 October 1996; received in revised form 3 February 1997; accepted 25 July 1997

Abstract
Higher plants are the source of a vast array of biochemicals which are used as drugs, pesticides, flavourings and
fragrances. For some of these compounds, plant cell culture can provide a potential production alternative to
traditional cultivation methods or chemical synthesis routes. Many systems have been patented and the last 20 years
have seen considerable industrial and academic interest in the development of large scale cultures to produce
pharmaceutically active, high value substances. However, the industrial application of plant cell suspension cultures
has, to date, been limited. Commercialisation has essentially been impeded by economic feasibility, arising from both
biological and engineering considerations. This paper reviews the commercial development of the technology to date
and focuses on the impact of specific engineering-related factors, in particular, the shear sensitivity of plant cell
suspension cultures. Evidence of sensitivity to hydrodynamic shear in bioreactors has generally been attributed to the
physical characteristics of the suspended cells. Recent studies indicate that shear sensitivity may not be as important,
in some cases, as initially anticipated. 1997 Elsevier Science B.V.
Keywords: Plant cells; Shear sensitivity; Bioreactor; Scale-up

1. Introduction
Higher plants are recognised as important
sources of a wide range of biochemicals, used as
drugs, pesticides, flavourings and fragrances. Tra* Corresponding author. Tel.: + 353 1 7045584; fax: + 353
1 7045412; e-mail: kieranp@ccmail.dcu.ie

ditionally, these substances have been extracted

from naturally grown whole plants. On a commercial basis, this approach involves large-scale
crop cultivation (e.g. alkaloids from Catharanthus
roseus). Many plant products can now be produced by chemical synthesis, which can be a more
reliable, consistent and cost-effective method.
Plant cell culture provides an alternative ap-

0168-1656/97/$17.00 1997 Elsevier Science B.V. All rights reserved.

PII S 0 1 6 8 - 1 6 5 6 ( 9 7 ) 0 0 1 6 3 - 6

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P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 3952

proach, which may be attractive under certain

circumstances: if, for example, the source plant is
difficult to cultivate, has a long cultivation period
or has a low metabolite yield; if chemical synthesis has not been achieved or if it is technically
problematic. Metabolite yield by the cell culture
may significantly exceed that observed in the parent plant. Thus, using this technology, the
metabolite can be produced under controlled and
reproducible conditions, independent of geographical and climatic factors.
The anti-cancer drug Taxol (a registered trademark of Bristol-Myers Squibb) is a very important example of a potential candidate for
production by cell culture methods. Taxol was
originally isolated from the bark of the Pacific
Yew tree, Taxus bre6ifolia. This slow growing tree
is principally found in the Pacific North-West. To
obtain 1 kg of Taxol requires the bark of more
than 1000 trees, each up to 100 years old. An
alternative approach to production is essential if
Taxol supplies are to be assured and if the
Pacific Yew population is not to be destroyed.
Total synthesis of Taxol has recently been reported (Holton et al., 1994, Nicolaou et al., 1994)
but is not economically sustainable on a large
scale and Bristol-Myers Squibb currently employs
a semi-synthetic process for the production of
Taxol, involving taxane precursors extracted
from various yew species. There is also preliminary evidence to suggest that fungi growing on
the Pacific Yew can produce both Taxol and
other taxanes (Stierle et al., 1995). If the biosynthetic pathway for Taxol can be fully elucidated,
genetically engineered synthesis may be possible.
Plant cell culture may yet provide another production route (Fett-Neto and DiCosmo, 1996;
Seki et al., 1997).
From an engineering perspective, cell suspension culture has more immediate potential for
industrial application than plant tissue or organ
cultures, due to the extensive body of expertise
which has been amassed for the treatment of
submerged microbial cultures. While tissue and
root cultures (Flores and Curtis, 1992) offer genetic stability as well as, in some instances, superior metabolic performance over suspension
cultures of the same lines, the development of

appropriate reactors and processing techniques

for these systems will involve enormous de novo
investment. Accordingly, most research effort has
been directed towards the commercialisation of
plant cell suspension culture. The purpose of this
paper is to provide an overview of the commercial
development of plant cell suspension culture technology to date and to focus on a limited number
of engineering issues, specifically hydrodynamic
shear sensitivity, which have hampered its exploitation on an industrial scale.

2. Commercial development
That plant cell culture offers the tantalising, but
as yet largely unattained prospect of long-term
commercial success, is evidenced in many recent
reviews concerning the technology and strategies
for its optimisation (Zenk, 1991; Verpoorte et al.,
1991; Scragg, 1992, 1995; Buitelaar and Tramper,
1992; Kreis, 1993; Shuler, 1993; Su, 1995; DiCosmo and Misawa, 1995; Dornenburg and
Knorr, 1995; Zhong et al., 1995; Schlatmann et
al., 1996). Despite its enormous potential, industrial processes involving plant cell cultures have
been limited to a handful of applications, including the production of shikonin from Lithospermum erythrorhizon (Fujita, 1988), berberine from
Coptis japonica (Fujita and Tabata, 1987) and
ginsenosides from Panax ginseng (Ushiyama,
1991). Many other processes have been investigated and patented but, to date, few have proven
to be economically viable. Plant cell cultures have
predominantly been valued as a source of naturally occurring secondary metabolites. However,
they can also be used for biotransformations such
as the glucosylation of hydroquinone to arbutin
by Rau6olfia serpentina (Lutterbach and Stockigt,
1992). In Japan, workers at Shiseido have alternatively employed C. roseus for the production of
arbutin by a similar process (Yokoyama and
Yanagi, 1991; Inomata et al., 1991). As a source
of enzymes for the genetically engineered synthesis of natural products (Scott, 1994), they offer
great promise. And large-scale processes for the
use of cell cultures as a source of biomass (Hashimoto and Azechi, 1988) have been investigated.

P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 3952

Reviews on the potential of plant systems for the

production of valuable products by these routes
are presented by Parr (1989), Pras (1992), Alfermann and Petersen (1995) and Stockigt et al.
(1995).
The recent literature and patent libraries (Verpoorte et al., 1991; Su, 1995) reveal the predominance of Japanese companies in the search for
commercial applications of plant culture technology. Spearheaded by the company Plant Cell Culture Technology, research in Japan has received
strong support from both government and industry (Misawa, 1991; Hara, 1996) and has produced
almost all of the commercialised processes. In the
USA and Europe, however, there have been fewer
commercial applications and most research effort
has been concentrated on a limited number of
high value products, including Taxol and the
indole alkaloids. Phyton Catalytic recently filed a
patent for the production of Taxol from highyielding cell cultures of Taxus chinensis (Bringi et
al., 1995). It has been reported (Anonymous,
1994) that ESCAgenetics, which filed a patent for
the production of vanillin using tissue culture
(Knuth and Sahai, 1991), was also involved in
Taxol production scale-up trials.
Limited commercialisation of the technology to
date can essentially be attributed to matters of
economic feasibility which, in turn, derive largely
from a combination of biological and engineering
factors. Plant cell culture, on an industrial scale, is
an inherently capital intensive process. Product
concentrations and productivities are typically
low. On this basis, its use can only be justified if
it offers an economic advantage over chemical
synthesis or traditional extraction processes, or if
no other alternative production route exists.
Shuler (1993) predicted that pharmaceuticals produced naturally by slow-growing, woody plants
(e.g. Taxol) are the most likely candidates for
development. In comprehensive reviews on the
production of alkaloids, Verpoorte et al. (1991,
1993) compared the results of a number of economic evaluation studies for the production of
alkaloids by cell culture. Their analysis confirmed
that while product price would be high and process optimisation essential, cell culture processes
could be feasible for speciality chemicals. How-

41

ever, the calculated product prices vary substantially with the choice of production strategy (e.g.
batch culture with product retained intracellularly; spontaneous or induced product release,
etc.). However, economic feasibility studies are
unanimous in acknowledging productivity as a
limiting factor. For example, Drapeau et al.
(1987) estimated that a 40-fold increase in the
ajmalicine productivity of C. roseus would be
required to justify the production of this compound by cell culture methods.
With regard to secondary metabolites, the productivity of plant cell suspensions is of the order
of 10 2 10 1 g l 1 per day (Scragg, 1995). Due
to the fact that productivity depends on product
yield, the organism growth rate and prevailing
biomass levels, it is clear that there is a role for
both biologists and engineers in improving system
performance. The question of economic feasibility
could be largely resolved by the development of
stable, high-yielding strains (Berlin, 1988) and the
identification of optimal cultivation conditions.
While the high biomass levels required for economic viability necessarily limit the volume of free
medium available, in an ideal system the cells
would actively secrete the product into the suspending process fluid (Buitelaar and Tramper,
1992), thereby simplifying downstream processing. However, to achieve these objectives, a fuller
understanding of the biosynthetic pathways is essential, in addition to a clearer picture of the
interactions between growth kinetics, system morphology, cellcell interaction and product synthesis.

3. Engineering considerations
From an engineering perspective, the primary
challenges lie in the area of process scale-up. Plant
cell suspensions can now be almost routinely cultivated in small-scale configurations. Moreover, in
addition to the commercial processes mentioned
earlier, there are a number of examples of largescale, albeit non-commercial systems. Nicotiana
tabacum cultures have been successfully cultivated
in a 20 m3 stirred tank reactor (Noguchi et al.,
1977). Westphal (1990) reported on long-term cul-

P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 3952

42

Table 1
Summary of biocatalyst characteristics
Biocatalyst

Shape

Size (mm)

Cell wall

Aggregates

Doubling time (h)

Qao (mmol l1 h1)

Plant cells

Spherical
Cylindrical

l: 100500
d: 2050

Yes

Yes

20 100

101

Animal cells

Spherical

d: 1020

No

No

20

101

Bacteria

Spherical
Cylindrical
Spiral

d: B1
l: B5

Yes

Yes/No

Yeasts

Spherical

d: 510

Yes

Yes/No

10

102

Moulds

Mycelial

d: 510
l: B100

Yes

Yes/No

10 20

102

0.5 10

103

Characteristic oxygen consumption rate for biocatalyst in liquid culture.

tivation of a number of lines in 50 m3 vessels.

However, in many cases, scale-up has been accompanied by a reduction in system productivity
(Schiel and Berlin, 1987; Ikeda, 1991; Taticek et
al., 1991), variously attributed to mixing and mass
transfer problems in the characteristically nonNewtonian broths, which exhibit varying degrees
of aggregation and which, ideally, have high
biomass concentrations. Product recovery is complicated, both technically and economically, by
the fact that most systems retain the product of
interest intracellularly, in the vacuole, thereby necessitating either cell lysis or non-destructive permeabilisation.

3.1. System characteristics and bioreactor design

A number of reviews have dealt with the choice
of bioreactors for plant cell suspension culture
(Kargi and Rosenberg, 1987; Payne et al., 1987;
Panda et al., 1989; Doran, 1993) and most emphasis has been placed on modifications of the
conventional stirred tank reactor (STR), with
bubble aeration, employing a variety of impeller
designs. However, equipment normally employed
for microbial cultivation may not be immediately
applicable to plant cells, due to differences between the characteristics of both individual cells
and suspensions of the respective systems. By way
of illustration, a summary of some of the physical
properties of biological systems is presented in
Table 1.

3.1.1. Aggregation
Plant cells are significantly larger and slower
growing than most microbial organisms. Individual plant cells have a typically characteristic
length of the order of 101 102 mm and may be
spherical to cylindrical in shape. Aggregation is
common, largely due to the failure of cells to
separate after division, although the secretion of
extracellular polysaccharides (ECP), particularly
in the later stages of batch growth, may contribute to increased aggregation (Taticek et al.,
1991). The aggregation phenomenon has been
used in the development of self-immobilisation
methods (Prenosil et al., 1987; Hegglin et al.,
1990). Aggregates, comprising up to 102 cells, may
be many millimetres in diameter (Tanaka, 1982)
and typically exhibit a tendency to settle. Aggregation patterns, variously studied using image
analysis (Kieran et al., 1995) and sieving (Mavituna and Park, 1987) techniques, vary significantly between cell lines and also as a
consequence of culture age and cultivation conditions. For example, N. tabacum, which is frequently
described
as
highly
aggregated
(Hashimoto and Azechi, 1988; Hooker et al.,
1989; 1990) exists, under certain conditions as
unbranched chains of up to 50 cells (DeJong et
al., 1967). Zenk et al. (1975) reported that 60% of
a Morinda citrifolia culture existed as single cells
or cells of two chains, whereas, in a study by
Kieran et al. (1993) the corresponding figure lay
between approximately 10% (lag and stationary

P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 3952

phases) and 50% (exponential phase). Wagner and

Vogelmann (1977) observed a change in the morphology of suspensions of C. roseus from pelleted
to single cells on scale-up from shake flasks to an
air-lift reactor. These examples emphasise the importance of identifying morphological trends under actual growth conditions and with due
reference to culture age. Deviations from expected
aggregate distributions may be indicative of culture variations in response to environmental factors. Although the role of cell cell interactions in
undifferentiated systems has yet to be conclusively
established, Shuler (1993) discusses evidence to
suggest that metabolite productivity may be significantly influenced by the degree of cellular association and may, therefore, be affected by
variations in aggregation patterns arising on
scale-up.

3.1.2. Rheology
Aggregation, aggregate interactions, high
biomass concentrations (e.g. up to 70 g l 1 on a
dry weight basis (Matsubara and Fujita, 1991))
and, in some cases, ECP secretions, result in high
whole-broth viscosities for plant cell suspensions.
There have been no comprehensive on-line rheological studies. Data have been collected for a
variety of plant cell systems using conventional
viscometers, although, as with other microbial
suspensions, care must be taken to avoid sedimentation effects (Scragg et al., 1986) and/or aggregate disruption (Rosenberg, 1987). Using the
concept of the apparent viscosity of a fluid (Metzner and Otto, 1957) rotational devices fitted with
purpose-built impellers have also been used for
the rheological characterisation of plant cell suspensions: for example, helical ribbon impellers,
designed to satisfy the need for good suspension
mixing at the widest possible range of laminar
flow conditions (Jolicouer et al., 1992; Kieran,
1993). A summary of relevant studies is presented
in Table 2 and it is apparent that the majority of
suspension cultures investigated exhibit non-Newtonian, shear-thinning characteristics. Many systems also show evidence of a yield stress.
Thixotropic behaviour has been observed, although only in isolated samples (Wagner and

43

Vogelmann, 1977). In common with many microbial suspensions, the apparent viscosity is found
to be strongly dependent on biomass concentration (Tanaka, 1982; Zhong et al., 1992a; Kieran,
1993), although the data reported in the literature
do not, in general, refer to the high density suspensions required for economically viable largescale processing. The influence of the morphology
of the suspended cells and/or aggregates on the
apparent viscosity of the suspension requires further investigation (Zhong et al., 1992a; Curtis and
Emery, 1993); it should also be noted that the use
of biomass concentration (on a dry weight basis)
as a correlating factor for viscosity can mask
effects attributable to variations in individual cell
size and water content over the course of the
growth cycle.

3.1.3. Oxygen and aeration effects

The oxygen requirements of plant cells are comparatively modest. Specific oxygen consumption
rates, on a dry weight basis, are of the order of
10 6 g g 1 per second, (Bond et al., 1988;
Dubuis et al., 1995; Ho et al., 1995). However,
high cell densities and high fluid viscosities can
reduce oxygen mass transfer efficiencies in bioreactor systems. Although critical dissolved oxygen
concentrations of approximately 1520% air saturation are commonly quoted for plant cell suspension cultures (Payne et al., 1992), the critical value
for cell growth may be significantly lower than
that for metabolite synthesis and recent studies
(Schlatmann et al., 1995) point to the importance
of dissolved oxygen concentration for metabolite
productivity.
Studies of the effects of aeration on plant suspension cultures have focused largely on the influence of kLa, the system mass transfer coefficient,
in which the combined effects of aeration and
agitation are inextricably linked. Kato et al.
(1975), Tanaka (1981), Smart and Fowler (1981)
and Leckie et al. (1991) all investigated the effect
of initial mass transfer coefficients on system performance in a variety of bioreactor configurations. Although the results are system specific, it
was generally concluded that for each system, a
lower limiting value of kLa exists, below which the

P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 3952

44

Table 2
Rheological characterisation of plant cell suspension cultures
System

Biomass concentrationa (g l1)

Characterisationb

Viscometric device

Reference

Papa6er somniferum
Nicotiana
tabacum
Batch cultivation
Semi-continuous
Nicotiana
tabacum
Perilla frutescens

B14.5

Newtonian

Brookfield; modified Stormer concentric

cylinder
Brookfield; modified Stormer concentric
cylinder

Curtis and Emery

(1993)
Curtis and Emery
(1993)

= 9.0

Pseudoplastic,
n= 0.6
Newtonian

Datura stramonium

=7.9
513

Brookfield-type

Kato et al. (1978)

B20

Pseudoplastic,
n :0.7
Bingham plastic

Brookfield-type

5450 (fresh weight)

Casson plastic

Modified Weissenberg rheogonimeter

Zhong et al.
(1992a)
Ballica et al.
(1992)
Ballica and Ryu
(1993)
Kieran (1993)

Contraves rheomat; Brookfield

Morinda citrifolia

5450 (fresh weight)

Catharanthus
527
roseus
Cudriana tricuspi- 515
data
Catharantus
roseus
Nicotiana
tabacum
a
b

Pseudoplastic,
n: 0.8
Pseudoplastic,
0.1BnB0.9
Pseudoplastic

Double-helical ribbon impeller

Helical ribbon impeller

Jolicouer et al.
(1992)
Tanaka (1982)

n: 0.53

Dry weight basis unless otherwise indicated.

n, flow behaviour index.

culture is inhibited; reduced productivity observed at higher gassing rates is variously attributed to the stripping of CO2 and essential
volatiles from the system (Kato et al., 1975;
Ducos and Pareilleux, 1986) or shear-related effects (Ballica and Ryu, 1993). Using a gas recirculation bioreactor for scale-up studies
involving C. roseus, Schlatmann et al. (1993)
confirmed the importance of dissolved gaseous
components for system performance and concluded (Schlatmann et al., 1994) that loss of an
unidentified essential volatile factor was responsible for the reduced ajmalicine synthesis observed on scale-up from a shake flask to an
aerated bioreactor.
Aeration of bioreactors can lead to foaming
and in plant cell suspension cultures this prob-

lem can be particularly severe (Zhong et al.,

1992b). Although there have been few comprehensive studies of this phenomenon and all
have been limited to laboratory or pilot scale
systems, foaming has typically been correlated
with aeration rates and extracellular protein
concentrations (Wongasmuth and Doran, 1994).
However, the contribution of extracellular
polysaccharides and other medium components
to foaming potential or foam stability has yet
to be established. A number of antifoams have
been used to control foaming in plant cell suspensions (Smart and Fowler, 1981; Zhong et
al., 1992b; Wongasmuth and Doran, 1994; Li
et al., 1995) resulting, in some cases (Smart and
Fowler, 1981; Wongasmuth and Doran, 1994)
in reduced system productivities.

P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 3952

3.1.4. Bioreactor design

As with microbial systems, approaches to overcoming suspension, mass transfer and mixing-related problems typically include improved reactor
design and, more commonly, increasing agitation
and aeration intensity. However, solutions must
be achieved without concomitant negative shearrelated effects. In STRs, large, slow-moving impellers often provide good mixing at relatively low
rotational speeds. Oxygen transfer may be limiting, due to poor bubble dispersion. However,
Jolicouer et al. (1992) reported on the successful
use of a double helical ribbon impeller, in an 11 l
surface-baffled vessel used for the cultivation of
C. roseus cultures. Bubble columns and air-lift
loop reactors offer the promise of a low-shear
environment (Moo-Young and Chisti, 1988) and
they have been used by a number of workers for
plant cell suspension systems (Smart and Fowler,
1981; Hegarty et al., 1986; Fulzele and Heble,
1994; Matsushita et al., 1994). Here again, performance is limited by mixing efficiency (Doran,
1993) which is reduced at the broth viscosities
associated with the high biomass levels necessary
for an economically viable process.
There have been many reports on the development of novel bioreactors for plant cell suspension culture. For example, rotating drum reactors
(RDR) have been used for the cultivation of C.
roseus (Tanaka et al., 1983), N. tabacum
(Shibasaki et al., 1992) and L. erythrorhizon
(Takahashi and Fujita, 1991). In the latter case,
the choice of an RDR in preference to either an
air-lift reactor or a paddle-agitated STR was
based on its superior performance in terms of
suspension homogeneity, low-shear environment
and reduced wall growth. Fluidised-bed reactors
have been proposed for industrial scale use
(Dubuis et al., 1993, 1995; Khlebnikov et al.,
1995), offering the possibility of perfusion operation with a facility for cell harvesting, a low-shear
environment and increased cell cell interaction.
Light irradiation has been shown to have a stimulating effect on the secondary metabolism of some
systems (Zhong et al., 1991; Furusaki et al., 1993),
but its integration into the operation of large-scale
bioreactors is problematic.

45

3.1.5. Shear sensiti6ity in plant cell suspension

cultures
The hydrodynamic shear sensitivity of biological systems, including prokaryotic and eukaryotic
suspensions and enzyme solutions, has attracted
considerable research attention in recent years
and has been comprehensively reviewed by a
number of authors (Thomas, 1990; Markl et al.,
1991; Merchuk, 1992; Hua et al., 1993; Joshi et
al., 1996). These systems encompass a range of
particle sizes, as well as varying degrees of structural and metabolic complexity, all of which impact on their sensitivity to a given shear
environment and on the extent of their responses.
While few submerged systems are adversely affected by the hydrodynamic environment associated with laboratory maintenance conditions,
process scale-up and the cultivation of high-density suspensions increase the mass transfer requirements of the system and accordingly, with
conventional processing equipment, the intensity
of the aeration and agitation conditions.
The response of a biological system to any
hydrodynamic environment depends on the duration and intensity of the applied conditions as well
as on the physiological characteristics of the system itself. The response may be positive (e.g. an
increase in biomass yield or secondary metabolite
synthesis). Investigations of shear sensitive systems, however, generally focus on the negative or
damaging effects associated with the hydrodynamic conditions encountered in process equipment. System response to shear may be measured
and assessed in a number of ways. Techniques
employed for plant cell systems are summarised in
Table 3. With a view to system scale-up, it is
interesting to note how few of the measurement
techniques listed lend themselves to on-line applications. In a number of studies (Dunlop et al.,
1994; Takeda et al., 1994), it is emphasised that
the quantitative evaluation of shear-related effects, in particular of sub-lytic effects, depends
crucially on the choice of damage indicator, which
frequently precludes the direct comparison of results collected from different studies.
Shear studies on biological systems, in general,
can be broadly divided into two categories,
classified in terms of the prevailing shear environ-

46

P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 3952

Table 3
Methods used for the assessment of shear-related effects in plant cell suspension cultures
System response

Parameter measured

Reference

Reduction in viability

Growth rate
Regrowth potential
Membrane integrity
Dye exclusion
Dual isotope labelling
Dielectric permitivity

Rosenberg (1987), Meijer et al. (1993)

Rosenberg (1987), Scragg et al. (1988)
Takeda et al. (1994), Kieran et al. (1995)
Parr et al. (1984); Kieran (1993)
Markx et al. (1991)

Release of intracellular components

pH variation
Protein release
Total organic carbon
Secondary metabolite release

Wagner and Vogelmann (1977)

Meijer et al. (1993)
Meijer et al. (1993)
Hooker et al. (1989)

Change in metabolism

Oxygen uptake rate

Respiration activity (TTC
reduction)
ATP concentration
Metabolite productivity
Cell wall composition

Ho et al. (1995)
Rosenberg (1987), Takeda et al. (1994), Zhong et al.
(1994)
Takeda et al. (1994)
Hooker et al. (1989); Zhong et al. (1994)
Tanaka et al. (1988)

Changes in morphology and/or

aggregation patterns

Aggregate size/shape

Takeda et al. (1994), Kieran et al. (1995)

Expansion index

Zhong et al. (1994)

ment and the duration of cell exposure. In the first

category, the biological suspension is exposed to
shear forces under growth conditions, for the
duration of cultivation or a significant portion
thereof (Scragg et al., 1988; Meijer et al., 1994;
Takeda et al., 1994; Zhong et al., 1994; Kieran et
al., 1995). Although these studies tend to be
highly system-specific, due to the variety of bioreactors employed, it is arguable that analysis under
actual growth conditions, or in a scaled down
version of a production bioreactor, offers the
greatest potential for successful scale-up of results
(Meijer et al., 1994; Ho et al., 1995). The hydrodynamic environment is generally regulated by
changing the rate or method of agitation and/or
aeration. Due to the difficulties involved in quantifying the levels of shear to which an organism is
exposed in the turbulent field of an agitated bioreactor, the intensity of the environment has been
generally related to impeller speed or power input.
The benefits of examining the shear sensitivity
of a system under actual or proposed operating
conditions are not to be underestimated. As the
apparatus is normally designed for sterile opera-

tion, the experimental exposure time is limited

only by the kinetics of the system itself and by the
ability of the organism to survive under the prevailing conditions. Use of chemostat culture (Meijer et al., 1993) facilitates the investigation of
long-term effects, but does not, in general, reflect
actual production conditions, where batch processing is most commonly employed and, moreover, may be less appropriate for systems yielding
non-growth associated metabolites.
In the second type of study, cells are exposed to
well-defined, laminar or turbulent flow conditions
(e.g. in couette, capillary and submerged jet
devices), for short periods of time, generally under
non-growth conditions. Studies involving plant
cell suspension cultures are summarised in Table
4. An exponential decay model is commonly employed to describe viability loss with increasing
exposure time to the imposed shearing conditions;
the resultant death or decay rate is used to quantify system response to a given hydrodynamic
environment (Kieran et al., 1995). Frequently, the
concept of a critical shear stress is employed. For
example, on the basis of system response to lami-

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47

Table 4
Defined flow-field shear experiments using plant cell suspension cultures
Cell suspension

Apparatus

Flow regime

Damage indicator

Reference

Morinda citrifolia

Recirculating flow capillary

Laminar/turbulent

Kieran et al. (1995)

Submerged jet

Turbulent

Dye exclusion
Morphology
Dye exclusion
Morphology

Daucus carota

Couette viscometer

Laminar

Morphology
Regrowth
Cell lysis

Nicotiana
tabacum

Couette-type

Transitional/turbulent

Viability
Hooker et al. (1989)
Cell lysis
Metabolite production

Perilla frutescens Couette-type

nar flow conditions in a viscometric device, Vogelmann et al. (1978) suggested a critical shear stress
of between 80 and 200 N m 2 for M. citrifolia;
using regrowth potential as an indicator of system
response, a critical shear stress of 50 N m 2 was
identified for suspensions of Daucus carota
(Rosenberg, 1987). Recent studies (Dunlop and
Namdev, 1993; Kieran et al., 1995; MacLoughlin
et al., 1997) have pointed to the use of energy
dissipation as a correlating factor for shear-related damage. However, given the variety of aggregate morphologies exhibited by plant cell
systems, no single mechanism for cell damage has
been conclusively identified. Because these devices
are generally operated under non-sterile conditions, system response is most frequently monitored in terms of loss of viability and accordingly,
more subtle, non-lytic effects may be overlooked
(Namdev and Dunlop, 1995).
There are a number of important conclusions to
be drawn from these studies which have significant consequences for the success of commercial
systems in conventional bioreactors. Interestingly,
it appears that plant cell suspensions are more
robust than initially anticipated and that significant losses of viability are not, in general, to be
expected under normal operating conditions in a
conventional STR. However, other less dramatic
shear-related effects may be observed, including

Respiration activity

MacLoughlin et al. (1997)

Rosenberg (1987)

Zhong et al. (1995)

reductions in metabolite yield (Hooker et al.,

1990) and biomass productivity (Ho et al., 1995).
Metabolic responses may be related to changes in
cellcell interactions effected by shear-induced aggregate disruption. System response may vary significantly between cell lines, and may also depend
on culture age and cultivation history. For example, Wagner and Vogelmann (1977) reported evidence of shear sensitivity in cultures of C. roseus.
In subsequent studies by Meijer et al. (1993), C.
roseus was found to be considerably more robust
than cultures of Cinchona robusta and Tabernaemontana di6aricata. Namdev and Dunlop (1995)
recently highlighted limitations of shear-related
studies, as conducted to date and proposed a
model for a more systematic and holistic evaluation of the response of plant cell systems to
hydrodynamic environments, focusing on calcium
transport, stress protein expression, osmo-regulation and aggregation. Overall, however, it appears
as if research effort might be more profitably
devoted to the development of shear-resistant cell
lines, rather than low-shear bioreactors.

4. Conclusions
This paper has considered only a limited number of engineering-related issues which impinge

48

P.M. Kieran et al. / Journal of Biotechnology 59 (1997) 3952

upon the commercialisation of plant cell suspension technology. The overall focus of related research is the achievement of large-scale cultivation
of high-yielding cell lines and efficient product
recovery, through the integration of biologicallyand engineering-based approaches. Process modelling has been successfully used as a tool for the
design, analysis and optimisation of many biological systems, including plant cell suspension cultures (Hooker and Lee, 1992; van Gulik et al.,
1993), but there is scope for development of models which take account of the fact that suspensions may not be homogeneous with respect to
metabolite synthesis. In the area of process control, a large-scale operation of plant systems is
hampered by difficulties associated with the online analysis of parameters such as cell or aggregate size distributions which may play an
important role in metabolite productivity. Different operating strategies (e.g. batch, two-stage,
continuous, etc.) may be required for cell lines
exhibiting different metabolite synthesis kinetics
(e.g. growth associated and non-growth associated) and which either store the product of interest intracellularly or, preferably, secrete it into the
suspending fluid. Non-lethal methods for inducing
product release and the use of two-phase systems
(Buitelaar and Tramper, 1992) can facilitate continuous processing without loss of biomass. The
product can be concentrated and by removing
feed-back inhibition effects, productivity may be
improved. Immobilisation (Hulst and Tramper,
1989) offers the possibility of a low-shear environment, enhanced cell cell contact, biomass re-use
and, if product secretion can be achieved, a facility for product removal. Some of these advantages have already been demonstrated using
self-immobilised plant cell systems in fluidised-bed
reactors (Dubuis et al., 1993; Khlebnikov et al.,
1995). Despite the obvious attractions of these
integrated processing approaches, problems of
both economic and technical feasibility must be
resolved before their application on an industrial
scale can be considered. Through concerted research efforts in the areas discussed in this paper
and in those highlighted above, wide-scale commercialisation of plant cell suspension technology
may soon be feasible.

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