Beruflich Dokumente
Kultur Dokumente
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
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
601
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
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
602
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
(5)
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.
(8)
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
2Cf
(1 K 2 )
2
=
n[(1/K)2/n 1]
(7)
KR =
603
2/n
R
r
(12)
604
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.
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 )
0.43
3.9
9.8
0.98
0.012
0.49
0.020
a
b
0.03
0.2 108
0.5 1010
0.04
0.001
0.02
0.001
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%.
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.
606
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
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
Acknowledgments
The authors acknowledge and appreciate the financial
support received from the National Aeronautics and Space
Administration (NAG 9-826).
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