Enzyme and Microbial Technology 31 (2002) 600608

Prolonged shearing of insect cells in a couette bioreactor

Kim C. OConnor a,b, , Nancy L. Cowger a,b,1 , Daniel C.R. De Kee a , Ray P. Schwarz c
a

Chemical Engineering Department, Tulane University, New Orleans, LA 70118, USA

Interdisciplinary Graduate Program in Molecular and Cellular Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA
c Synthecon, Houston, TX 77054, USA
Received 20 August 2001; received in revised form 15 April 2002; accepted 21 April 2002

Abstract
Viscometers have had a prominent role in the study of hydrodynamic damage to cell cultures. A Couette bioreactor overcomes stringent
time limits of previous viscometric research. At low shear levels, the vessel supported robust growth of Spodoptera frugiperda cells (>92%
viability) at 0.64 0.09 day1 to a maximum cell density of 6.1 106 cells ml1 . The intrinsic rate of necrosis was 30+ times slower than
the specific growth rate. Cell death was bimodal, confirming recent reports of apoptosis in uninfected insect cells. Population dynamics
suggests that early apoptotic cell formation accelerated 100-fold over a month, surpassing the rate of necrosis by day 4. Early apoptosis
was the rate-limiting step in the apoptotic pathway and particularly sensitive to culture conditions. Accepted methods of estimating shear
exposure were revised to account for the pseudoplasticity of Couette cultures: power-law parameters m = 0.660 0.054 dyne s0.22 cm2
and n = 0.222 0.047. The maximum shear stress of 0.84 dynes cm2 was 70+ times the value predicted for a Newtonian fluid (1.0 cp
viscosity). Prolonged shearing in a Couette bioreactor will enable investigation of cumulative stress effects and the shear response of cell
processes with a long half-life.
2002 Elsevier Science Inc. All rights reserved.
Keywords: Insect cells; Couette bioreactor; Apoptosis; Necrosis; Viscosity; Shear

1. Introduction
Animal-cell cultivation is the source for a variety of
valuable therapeutic and diagnostic products, including
monoclonal antibodies and recombinant proteins [1,2]. For
commercial production, cells are often cultured on a large
scale and in suspension within stirred bioreactors. Agitation
suspends cells and enhances mass transport of nutrients and
wastes, but the mechanical stresses that it generates can be
detrimental to fragile animal cells. Excessive hydrodynamic
forces are a known cause of physical damage and death in
animal-cell culture [3]. Even at moderate levels, these forces
can significantly alter cell physiology [4]. By protecting
cells from hydrodynamic damage, the yield and activity of
cell products can be improved [5,6]. This necessitates an
understanding of the mechanisms of hydrodynamic damage
to cell culture.
Agitation exposes suspension cultures to complex flow
patterns in which the cell surface is deformed in different

Corresponding author. Tel.: +1-504-865-5740; fax: +1-504-865-6744.

E-mail address: koc@tulane.edu (K.C. OConnor).
1 Present address: StelSys, LLC., 1450 South Rolling Rd., Baltimore,
MD 21227, USA.

directions with varying degrees of severity and duration [3].

Viscometric devices provide a well-defined flow profile to
investigate the biological response elicited by flow-induced
deformation. Attachment-dependent cells have been sheared
in parallel-plate flow chambers to explore, for example, the
effects of fluid flow on gene expression in recombinant
Chinese hamster ovary cells [7]. For attachment-independent
cells, viscometers have been instrumental in characterizing
the influence of such variables as plasma membrane fluidity and the concentration of protective agents on the shear
sensitivity of hybridomas and insect cells [5,6,8].
The residence time for cell suspensions in bioreactors
is on the order of days or weeks, and some cellular processes, such as population dynamics [9], have a half-life
measured in hours or days. In addition, data in the literature suggest that the cellular response to shear may depend
on exposure time and, in particular, the cumulative work
done on the cells [1012]. While circulation of medium
through parallel-plate chambers facilitates long-term exposure of attachment-dependent cells to shear, this is not
the case for attachment-independent cells in conventional
viscometers. Without medium exchange or oxygenation,
shearing in these devices has been limited to a few hours at
most. An alternate design is required to investigate cellular

0141-0229/02/$ see front matter 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 1 3 7 - 0

K.C. OConnor et al. / Enzyme and Microbial Technology 31 (2002) 600608

601

processes with a long half-life and cumulative shear effects

over periods comparable to residence times in bioreactors.
The present study explores shearing cell suspensions
within a Couette bioreactor in the annular space between
two concentric cylinders rotating independently around a
horizontal axis. Maurice Couette originally designed this
configuration in 1890 [13]. Flow regimes generated by
Couette devices have been extensively characterized [14].
Several of the viscometers employed for short-term shearing
of cell suspensions had a Couette design [8,1012]. Flow
profiles within Couette bioreactors have been described
under quiescent and agitated conditions in the absence of
cells [15,16].
In this work, we explored whether a Couette bioreactor
can support cell growth during prolonged exposure to low
levels of shear. Insect cells were chosen for this study because of their popularity as production hosts for high yields
of recombinant proteins [2]. Our research group is interested in shear effects on population dynamics to develop
strategies to enhance culture longevity. This initial characterization of the Couette culture provides insight into cell
death mechanisms, particularly apoptosis. There is very little information available on the rheological properties of
animal-cell suspensions with which to characterize the shear
profile in the bioreactor. In contrast to previous viscometric
research with dilute suspensions [8], the more concentrated
insect-cell culture was highly pseudoplastic. A comprehensive Couette theory, which accounts for this non-Newtonian
behavior, is reviewed.
2. Materials and methods
2.1. Cell cultivation
IE1FB2 is an attachment-independent subclone of Sf9
Spodoptera frugiperda cells. Cultures were grown in suspension at 27 C and 95% relative humidity in Ex-Cell 401
medium at pH 6.5 containing 325 mg dl1 glucose (JRH
Biosciences, Lenexa, KS). Insect cells were sheared within
a Couette bioreactor (Synthecon, Inc., Houston, TX) in the
annular space between two concentric cylinders (Fig. 1). The
culture chamber had the following dimensions: length =
5.7 cm, diameter of the inner cylinder = 4.0 cm, and outer
cylinder diameter = 6.0 cm. The cylinders were separated
by a wide gap of 1 cm to minimize cell disturbance of
flow profile and had tapered ends to reduce secondary flow
[17]. Cultures were sheared by rotating the cylinders in
the same direction with a differential angular velocity of
3.0 0.3 rpm, with the inner cylinder rotating faster at 21
0.2 rpm. The bioreactor was inoculated with 2.0 0.1106
cells ml1 , operated in fed-batch mode and oxygenated via a
silicon membrane around the inner cylinder. The schedules
for medium replacement and oxygen flow rates were based
on those established by Cowger et al. [9] for insect-cell cultivation in quiescent environments (Table 1). Under these con-

Fig. 1. Photograph (A) of Couette bioreactor with sketches of reactor

components (B) and culture chamber (C). Nomenclature for select operating parameters is displayed in Part C.

ditions, glucose concentration, dissolved oxygen concentration, and pH were between 300150 mg dl1 , 15040 mmHg
and 6.45.6, respectively, and fell to their lower limits after
the onset of stationary phase.
Table 1
Scheduled medium replacement and oxygen flow rate for Couette
bioreactor
Day

Medium replacement (ml)

O2 flow rate (l h1 )

1
2
3
4
5
5
722

5
10
12
20
27
20/20a
25/25a

7
7
10
17
25
37
37

Medium replacement was twice per day.

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K.C. OConnor et al. / Enzyme and Microbial Technology 31 (2002) 600608

Cell concentrations were measured with a hemocytometer. The concentration of glucose and dissolved oxygen in
conditioned medium was measured with a Yellow Springs
Instrument Model 27 Industrial Analyzer (YSI glucose kit
2365, Yellow Springs, OH) and an 8730 flow-through oxygen electrode connected to an OM-4 oxygen meter (Microelectrodes, Inc., Londonderry, OH).
2.2. Rheology
Steady-shear viscosity of culture samples was measured
with a Dynamic Stress Rheometer SR-5000, employing RSI
Orchestrator analysis software (Rheometric Scientific, Inc.,
Piscataway, NJ). Two types of samples were prepared: a cell
suspension containing 5.5 0.5 106 cell ml1 in conditioned Ex-Cell 401 medium and the supernatant from centrifuged suspensions. The viscometer had a parallel-plate
design and was operated at 25 C with 40-mm diameter
plates. The gap between the plates was increased from 0.45
to 0.8 mm to confirm the absence of fluid slip. Instrument
standards (Rheometric Scientific) were used for spin correction and calibration. Viscosity was determined as a function
of shear rate, starting at 40 s1 and ending at 1.0 s1 .
2.3. Fluorescence microscopy
Apoptotic and necrotic cells in culture were detected with
fluorescence microscopy as described by Cowger et al. [9].
Briefly, cell samples were stained with the DNA-binding
dyes acridine orange and ethidium bromide at a concentration of 4 g ml1 each in PBS. The stained sample was immediately mounted on a slide and examined with an Olympus IX50 microscope (C2 Corp, Tamarac, FL) equipped
with an IX-RFA/S fluorescence illuminator and the following filter combinations: exciter filter, 470490 nm; dichromatic beamsplitter, 500 nm; and barrier filter, 515 nm. Digital images of fluorescence cells were captured with an Optronics DEI-750 digital camera (C2 Corp) and viewed with
Image-Pro Plus software (Media Cybernetics, Silver Spring,
MD). Cells were divided into four populations (viable, early
apoptotic, late apoptotic, and necrotic cells) based on their
staining pattern and morphology [18]. Viable and early apoptotic cells appear green; the former has a round, symmetric
nucleus, while the latter is smaller in size with a condensed
nucleus. Late apoptotic and necrotic cells fluoresce orange;
the DNA staining pattern remains condensed in late apoptotic cells and diffuse in swollen necrotic cells.
2.4. Kinetic model
Model equations describing population dynamics in cell
culture were solved numerically by the Eler method with
a time step of 0.05 days [9]. Numerical integration was performed with Microsoft Excel using the solver function as
a multi-variable minimization routine. Kinetic parameters

were determined by minimizing the sum of the normalized,

squared residual error between model predictions and experimental data.
2.5. Statistical analysis
Data are reported as mean standard deviation for triplicate cultures. Statistical analysis was performed with a
Students t-test. A probability (P) of 0.01 was the criterion
for significance.
3. Theory
3.1. Population dynamics
Insect cells undergo bimodal cell death by both apoptosis
and necrosis. We have developed a kinetic model to describe
population dynamics under these conditions [17]. Model
equations describe the accumulation of each cell population
as a balance between its rate of formation and depletion with
characteristic rate constants (k).


dX
= 0 X ks X 2 kEA X X dt kN X
(1)
dt


dCEA
= kEA X X dt kLA CEA
(2)
dt
dCLA
(3)
= kLA CEA kALys CLA
dt
dCN
(4)
= kN X kNLys CN
dt
where X is the concentration of viable cells; CN , necrotic
cells; CEA , early apoptotic cells; and CLA , late apoptotic
cells. Also, Lys denotes cell lysis; s, stationary phase; and
0 , the specific growth rate.
3.2. Viscosity
For non-Newtonian fluids, viscosity () can be considered
to be a function of the shear rate ( ). The power-law model
is frequently used to describe this dependence [19].
= m| |n1

(5)

The parameters m and n can be measured from a loglog

plot of versus . For Newtonian fluids, is independent
of with n equal to unity. The value for n can be confirmed
from the relationship between the torque (T) required for
rotation in a parallel-plate rheometer and evaluated at the
disk radius (R) [19]. For a power-law fluid:
log T
(6)
= n log R
3.3. Flow stability
Several flow regimes are observed in Couette devices
starting at low Reynolds numbers with laminar flow,

K.C. OConnor et al. / Enzyme and Microbial Technology 31 (2002) 600608

followed by Taylor vortex flow, and eventually turbulence

at high Reynolds numbers [14]. The stability of these flow
patterns has been investigated under various conditions
[14,20]. For two cylinders rotating in the same direction,
laminar flow is stable when
R R 2 > KR (KR)2

3.4. Shear
It is possible to predict at the inner wall of a Couette
device without prior assumption of a flow profile [21,22].
Let  represent the angular velocity difference between
the two cylinders.

This initial study focused on characterizing the population

dynamics of Couette cultures of S. frugiperda cells, rheology
of the cell suspension and shear profile within the culture
chamber. This was done at the lowest settings for cylinder
rotation required to produce a homogeneous cell suspension
as measured by drawing reproducible cell samples from the
reactor ports: KR = 21 rpm and R = 18 rpm. These rotational settings will serve as our low-shear control in future research. Cylinder rotation suspended the insect cells
in medium, and the velocity gradient between the inner and
outer cylinders sheared the culture.

(8)

The correction factor Cf has a value of unity for Newtonian

fluids and the following form for a power-law fluid:



K 2 1
2
1
1
Cf = 1 +
1 + ln K
1
3
n
2K 2

2
K 2 1
1 1
+
ln
K

1
+ ...
n
6K 2

(9)

Eqs. (8) and (9) were derived for an incompressible, isothermal fluid in a Couette device operating at steady state
without slip at the cylinder wall. These are reasonable
assumptions for our cell system.
Velocity and shear profiles in Couette devices can be readily estimated for laminar flow subject to the assumptions
described above [19]. The only non-vanishing velocity component is in the -direction (V ) and is a function of radial
distance (r).
 
 2/n
V
R 2/n

1
= KR +
(10)

2/n
r
r
K
[1 (1/K) ]
This solution is valid for a power-law fluid and satisfies the
boundary conditions V = R R at r = R and V = KR KR
at r = KR. Shear rate is defined in terms of a velocity
gradient.
 
d V
r = r
(11)
dr r
For a power-law fluid:
r

4. Results

4.1. Population dynamics

2Cf
(1 K 2 )

2
=
n[(1/K)2/n 1]

Shear stress ( r ) acting in the -direction on a fluid surface

of constant radial position r is the product of and r
n
 2 
2
R
r = m
(13)
r
n[(1/K)2/n 1]

(7)

Here, is the angular velocity; R, the radius of the outer

cylinder; and K, the ratio of the inner to outer cylinder radius.
Eq. (7) was derived by considering the forces acting on a
rotating fluid. Flow is stable when a fluid element displaced
by centrifugal force is returned to its initial position by a
radial pressure gradient [20].

KR =

603

 2/n
R
r

(12)

Population dynamics describe the rates at which animal

cells progress through growth, stationary and death phase. It
provides a comprehensive analysis of culture performance
from the prospective of viability. Upon inoculation with
2.0 0.1 106 cells ml1 , the Couette culture grew exponentially with a specific growth rate of 0 = 0.64 0.09
day1 (equivalent to a 26-h doubling time) without a detectable lag phase (Fig. 2A, Table 2). During exponential
phase, the viable cell population accounted for at least 92%
of the cells in culture. There was a prolonged stationary
phase starting 6 days after inoculation and lasting for another
6 days as the culture approached a maximum cell density of
6.1 0.2 106 cells ml1 .
Cell death was bimodal by both apoptosis and necrosis.
The intrinsic rate of necrosis, kN , was 0.019 0.001 day1 ,
more than 30 times less than 0 (Table 2). Necrotic cells
lysed more slowly than they were synthesized (kN X >
kNLys CN ), resulting in an accumulation of the necrotic cell
population to 25% of the culture by day 22 (Fig. 2A). On
the same day, the concentration of early apoptotic cells
was nearly half that of necrotic cells. In previous research,
we found that the intrinsic rate of early apoptosis was
time-dependent
and could be described mathematically as

kEA X dt [9]. This expression suggests that apoptotic cell
formation is dependent on the accumulation over time of
a factor synthesized by the cells. The relative rate of early
apoptosis to necrosis is plotted in Fig. 2B. Early apoptotic
cell formation was initially negligible and then rapidly
increased, exceeding kN by day 4. On day 22, the rate
of apoptosis was 10 times faster than the rate of necrosis. Throughout cultivation, early apoptotic cell formation
was the rate-limiting step
 as a cell progressed through the
apoptotic pathway: kEA X dt was less than kLA and kALys

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K.C. OConnor et al. / Enzyme and Microbial Technology 31 (2002) 600608

Table 2 includes kinetic constants describing the population dynamics of S. frugiperda cells in a high-aspect
rotating-wall vessel (HARV) [9]. While the HARV provides
a quiescent environment for cultivation, it does not have the
versatility of the Couette bioreactor in the extent to which
the shear profile can be defined and varied. A comparison of
population dynamics in the two reactors provides insight into
the sensitivity of the kinetic rate constants to changes in culture conditions. The largest differences in the two data sets
were for the values of kEA and kS . The rate at which a cell
progressed from a viable to early apoptotic state was faster
in the sheared Couette culture: kEA = 24.0 3.0 1010 ml
cell1 day2 , a factor of nearly 2.5 greater than for the
quiescent IIARV culture (P = 0.01). The rate constant kS
is inversely proportional to the maximum cell density and
was 2.3-fold larger for Couette cultures (P < 0.01). The
lower maximum cell density in the Couette bioreactor relative to the HARV may reflect a reduced rate of nutrient
supply to or waste removal from the reactor. Alternatively,
sheared cells may have elevated nutrient consumption or
waste production.
4.2. Viscosity

Fig. 2. Population dynamics of S. frugiperda cultures in Couette bioreactor as determined by fluorescence microscopy (symbols) and model simulation (curves). (A) Accumulation of viable (, ), early apoptotic
(, - - -), late apoptotic (, ::::::::) and necrotic ( , ) cells.
(B) Ratio of the intrinsic rate of early apoptosis to that of necrosis. The
reactor was inoculated with 2.0 0.1 106 cells ml1 and operated at
a differential angular velocity of 3.0 0.3 rpm with the inner cylinder
rotating at 21 0.2 rpm.

(Fig. 2B, Table 2). The accumulation of late apoptotic cells

to 12% of the Couette culture by day 22 indicates that
kLA CEA > kALys CLA . Since kLA = kALys , the accumulation
rate was governed by the relative concentration of early and
late apoptotic cells.

In previous viscometric research, cell suspensions have

been often approximated as Newtonian fluids for shear estimations [5,8,10,12,16]. This assumption was not valid for
the Couette culture. Viscosity measurements were performed
with 5.5 0.5 106 cells ml1 in conditioned Ex-Cell 401
medium. The cell suspensions contained 45% (v/v) solids
and were highly pseudoplastic, becoming more viscous at
lower shear rates (Fig. 3A). Specifically, viscosity increased
from nearly = 5 cp at r = 40 s1 to over 50 cp at
1.0 s1 . The loglog plot of versus r in Fig. 3A is linear for shear rates below 40 s1 , indicating that the cell
suspension can be modeled as a power-law fluid in this
region with characteristic parameters m = 0.660 0.054
dyne s0.22 cm2 and n = 0.222 0.047. The value for n
was confirmed from the slope of the loglog plot of torque
versus shear rate (Fig. 3B). Supernatant from the cell suspensions was less viscous by a factor of 2.5 on average at
40 s1 , and its pseudoplastic behavior was less pronounced:

Table 2
Comparison of rate constants for population dynamics of S. frugiperda cells in Couette bioreactor and HARV
Rate constant

Couette bioreactor

HARVa

0 (day1 )
kS (ml cell1 day1 )
kEA (ml cell1 day2 )
kLA (day1 )
kN (day1 )
kALys (day1 )
kNLys (day1 )

0.64 0.09 (P > 0.01)b

9.2 1.0 108 (P < 0.01)
24.0 3.0 1010 (P = 0.01)
1.1 0.1 (P > 0.05)
0.019 0.001 (P < 0.01)
1.1 0.1 (P < 0.01)
0.01 (P < 0.01)

0.43
3.9
9.8
0.98
0.012
0.49
0.020

a
b

Reported by Cowger et al. [9].

Probabilities from Students t-test, comparing rate constants for Couette and HARV cultures.

0.03
0.2 108
0.5 1010
0.04
0.001
0.02
0.001

K.C. OConnor et al. / Enzyme and Microbial Technology 31 (2002) 600608

605

Fig. 3. Conditioned Ex-Cell 401 medium exhibited pseudoplastic behavior. Dependence of viscosity (A) and torque (B) on shear rate for cell
suspensions () and supernatant () from S. frugiperda cultures containing 5.5 0.5 106 cells ml1 . Standard deviation for rheology data
was less than 5%.

m = 0.056 0.003 dyne s0.61 cm2 and n = 0.612 0.004

(Fig. 3A).
4.3. Velocity and shear profile
Our characterization of velocity and shear profiles in the
Couette bioreactor began with an analysis of flow stability.
According to Landaus and Liftshitzs criterion [20], flow
within the culture chamber was laminar for the operating
conditions presented here: KR = 21 rpm and R = 18 rpm
(Eq. (7)). In fact, the transition to Taylor vortex flow was
estimated to occur when the inner cylinder was rotating at
a significantly higher angular velocity: KR = 40 rpm
Fig. 4A compares the velocity profile for laminar flow
when the actual pseudoplastic behavior of the Couette culture was addressed with that predicted for an idealized Newtonian fluid. The latter is based on a commonly assumed
viscosity for culture medium: = 1.0 cp, the viscosity of
water [5,8,16]. Without slip, V /r equals the angular veloc-

Fig. 4. Estimates of fluid velocity (A), shear rate (B) and shear stress
(C) as a function of radial distance in the Couette bioreactor for cell
suspensions described in Fig. 3. () and a Newtonian fluid with viscosity
of 1.0 cp (). Cylinder rotation was the same as described for Fig. 2.

ity of the rotating cylinders at r = 2 cm (KR) and r = 3 cm

(R). Of the two cases considered, the estimated velocity of
the cell suspension was slightly slower by nearly 5% within
the annular space due to its higher viscosity and, therefore,
greater resistance to flow (Fig. 3A).
The velocity difference between the power-law and Newtonian fluid is reflected and magnified in the profiles for
shear rate and shear stress. Like the velocity profile, the estimated shear rate progressively decreased from the faster
to slower rotating cylinder in laminar flow (Fig. 4B). Since
r d(V /r)/dt (Eq. (11)), this reduction in shear rate
is more pronounced for the pseudoplastic cell suspension.

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K.C. OConnor et al. / Enzyme and Microbial Technology 31 (2002) 600608

At its maximum, r = 2.90 s1 for the Couette culture, a

2.5-fold increase over the estimated Newtonian shear rate.
These values are within 3% of those predicted without prior
assumption of a flow profile using Eqs. (8) and (9). At its
minimum, shear rate of the culture (r = 0.08 s1 ) was
substantially less than that of the Newtonian fluid (r =
0.50 s1 ). The net effect of the pseudoplasticity on viscosity
and shear rate was to elevate the shear stress by a factor of
more than 70 (Fig. 4C). For the Newtonian fluid, r 0.01
dynes cm2 throughout the annular space of the Couette
bioreactor. In contrast, the non-Newtonian shear stress was
estimated to range from 0.84 dynes cm2 at the inner cylinder to 0.37 dynes cm2 at the slower rotating outer cylinder.

5. Discussion
The Couette bioreactor supports prolonged shearing of
insect-cell suspensions. The low-shear control exhibited robust cell growth and high viability during exponential phase.
With respect to cell death, results suggest that early apoptotic cell formation accelerated during cultivation, was the
rate-limiting step in the apoptotic pathway and was particularly sensitive to culture conditions. Accepted methods of
estimating shear exposure were invalid for our system; the
cell suspension was pseudoplastic rather than Newtonian,
resulting in larger gradients in fluid velocity and shear rate
across the culture chamber, and substantially higher shear
stress.
5.1. Apoptosis
Knowledge of cell death mechanisms in culture is useful
in developing strategies to extend culture longevity and enhance the yield of cell products [23]. Our lab was the first
to quantify apoptotic S. frugiperda cells in uninfected cultures and to develop a kinetic model for this mode of cell
death [9]. Recently, Meneses-Acosta et al. [18] confirmed
our findings and observed that S. frugiperda cells die by an
atypic form of programmed cell death characterized, for example, by nonspecific DNA degradation. Without the traditional ladder pattern of DNA fragments, these insect cells
cannot be detected by agarose gel electrophoresis. The atypic
features of apoptotic S. frugiperda cells could explain why
they remained undetected for so long.
Kinetic results from the present study suggest that early
apoptotic cell formation was the rate-limiting step in the
apoptotic pathway and, as such, would be a likely target
for metabolic engineering to enhance culture longevity.
Consistent with these findings, transfection of hybridomas
with the human bcl-2 gene inhibited programmed cell death
during suboptimal culture conditions [23]. The bcl-2 protein is thought to antagonize protease activity, suppressing
apoptosis at an initial stage [24]. Our observation that the
rate-limiting step in the apoptotic pathway was also among
the most sensitive to changes in culture conditions may be

more than coincidental. It could be analogous to feedback

inhibition of an irreversible step in a metabolic pathway [25].
5.2. Pseudoplasticity
While the rheological properties of blood, fermentation
broths and plant-cell cultures have been extensively investigated [2628], there have been only a few reports of this
kind for animal-cell cultures. Concentrated HeLa cell suspensions in PBS are pseudoplastic with viscosities between
100 cp and 1000 poise [29]. A variety of fresh media are
reported to have an average viscosity of 1.01.5 cp and exhibit nearly Newtonian behavior [5]. Our study began to
explore the rheological properties of an animal-cell suspension under actual culture conditions. Like suspensions of
HeLa cells, S. frugiperda cultures were highly pseudoplastic. Probably this reflects a greater structure in the suspension and, therefore, more resistance to flow at lower shear
rates from particle aggregation and entanglement of macromolecules [30,31]. It follows that the pseudoplasticity of
cell-free culture medium in our study was less pronounced
than that of the insect-cell suspension. The viscosities in the
present study were 10- to 104 -fold lower than those for HeLa
cells since the insect-cell suspensions were less concentrated: 45% (v/v) versus 6090%. Plant-cell suspensions,
for example, also exhibit this dependence on cell concentration [27,32]. Relative to the fresh media described above,
our cell-free culture medium was at least 50% more viscous
most likely from cellular macromolecules actively secreted
in the medium during conditioning and passively released
during lysis. This has also been observed with cultures of
Beta vulgaris producing extracellular protein and polysaccharide [33].
In light of these findings, the assumption of Newtonian
behavior for animal-cell cultures is valid for very dilute cell
suspensions prepared with fresh medium. For conventional
viscometric studies in which the cell concentration is typically 0.5% (v/v) [8], these criteria can be readily satisfied.
Unfortunately the Newtonian approximation has been extended to more concentrated animal-cell suspensions [16],
and, as demonstrated here, can cause substantial error in
shear estimates. Temporal changes in culture viscosity require further investigation. It is likely that viscosity of the
cell suspension will become increasingly non-Newtonian as
cultivation proceeds from proliferation, conditioning and lysis. Moreover, a viscosity gradient may develop in the culture chamber. Particles migrate in a shear gradient toward
the low shear region [34]. In our Couette bioreactor, this corresponds to an accumulation of cells near the outer cylinder.
Cell migration would reduce viscosity at the inner cylinder
and have the opposite effect at the outer cylinder.
5.3. Error analysis
The Couette bioreactor was designed and operated to reduce several potential sources for error in shear estimates.

K.C. OConnor et al. / Enzyme and Microbial Technology 31 (2002) 600608

Eccentricity in cylinder alignment can produce flow instabilities, and there can be secondary flow at the ends of the inner
cylinder [35]. The wide gap and tapered ends in our Couette bioreactor would minimize these effects. In addition, the
viscous nature of our cell suspension would tend to stabilize
flow [20]. Slip would reduce shearing at the cylinder wall.
At the faster rotating inner cylinder where the shear rate was
highest, the silicon membrane has a textured surface inhibiting slip. With respect to reactor operation, cylinders were
rotated in the same direction because fluid flow is inherently
more stable than between counter-rotating cylinders [14].
Flow remains stable even at high Reynolds numbers when
only the outer cylinder is rotated [14]. Inner cylinder rotation reduces this transition but minimizes plugging of the
silicon membrane as cells migrate away from the faster rotating cylinder as described above [34]. In the present study,
the differential angular velocity between the two cylinders
was chosen to be well within the stable flow regime.
5.4. Applications
To date, viscometric studies of cell suspensions have been
limited by nutrient depletion to brief periods of elevated
shearing [5,6,8,1012]. Results from these experiments are
often cited to interpret culture performance without consideration for exposure time and have been used to establish shear thresholds above which cell damage initiates [11].
If the cumulative work done on sheared cells were to determine their performance, then brief exposure to elevated
shear would elicit the same cellular response as lower levels over longer times. Further, the shear threshold could be
lower than predicted by experiments lasting seconds. In support of these arguments, an inverse relationship has been
established between shear stress and exposure time for rupture of red blood cells [11] and viability of hybridomas [12]
for brief periods of shearing under 2 h. Dunlop et al. [10]
demonstrated that culture performance was a function of cumulative work done on cells for carrot plant cultures sheared
up to an hour. In addition, there have been several comparisons of cells grown in quiescent bioreactors, where the estimated shear stress was under 0.5 dyne cm2 , with cultures
from conventional vessels operating at approximately 11.5
dyne cm2 [36,37]. The quiescent cultures exhibited, for example, greater cell differentiation and extended longevity.

6. Conclusions
The Couette bioreactor overcomes stringent time limits
imposed on shearing animal-cell suspensions in traditional
viscometers. The longer exposure time will enable investigation of cumulative stress effects and the shear response of
slower cell processes, such as population dynamics. Concentrated cell suspensions from the Couette bioreactor exhibited
pseudoplastic behavior not evident in previous viscometric
research with dilute suspensions. Approximating our cell

607

culture as a Newtonian fluid grossly underestimated shear

stress. A comprehensive Couette theory is reviewed which is
applicable to dilute and concentrated cell suspensions alike.

Acknowledgments
The authors acknowledge and appreciate the financial
support received from the National Aeronautics and Space
Administration (NAG 9-826).

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