1 Nephrology Section, Tulane University Medical Center, Tulane Environmental Astrobiology Center, Center for BioEnvironmental Research, Louisiana Veterans Research Corporation, and Veterans Affairs Medical Center, New Orleans 70112; and 2 Screaming Jeanne's, Incorporated, New Orleans, Louisiana 70115
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ABSTRACT |
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Suspension culture remains a popular modality, which manipulates mechanical culture conditions to maintain the specialized features of cultured cells. The rotating-wall vessel is a suspension culture vessel optimized to produce laminar flow and minimize the mechanical stresses on cell aggregates in culture. This review summarizes the engineering principles, which allow optimal suspension culture conditions to be established, and the boundary conditions, which limit this process. We suggest that to minimize mechanical damage and optimize differentiation of cultured cells, suspension culture should be performed in a solid-body rotation Couette-flow, zero-headspace culture vessel such as the rotating-wall vessel. This provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis, characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. Optimization of suspension culture is achieved by applying three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Next, rotation in the rotating-wall vessel induces both Coriolis and centrifugal forces, directly dependent on terminal velocity and minimized as terminal velocity is minimized. Last, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. In the transduction of mechanical culture conditions into cellular effects, several lines of evidence support a role for multiple molecular mechanisms. These include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as protein kinase C. The discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and biological effects, emphasizing cellular processes important for the industrial production of biological pharmaceuticals and devices.
laminar flow; bioproducts; microcarriers; shear; gene expression
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SUSPENSION CULTURE |
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MOST IF NOT ALL DIFFERENTIATED cells derived from diverse tissue sources lose their specialized features and dedifferentiate when grown under traditional two-dimensional cell culture conditions (24, 25, 68). Suspension culture is the most popular means of preventing this problem and maintaining specialized features of cells. The rich and diverse array of available culture vessels for suspension culture makes selection of specific culture devices bewildering. There is a great deal of information available on the optimization of suspension culture by utilizing the engineering principles of fluid physics to minimize shear and turbulence (20, 24, 45, 47, 60, 61). However, for most biologists, translating this technical information into practical culture conditions is complex and counterintuitive.
Several recent reviews have summarized the use of suspension culture (2, 26), scale-up of suspension culture for industrial use (44), application to anchorage-dependent cells by the use of microcarriers (9, 20, 45), and automation of roller-bottle forms of suspension culture (37). Some earlier reviews have collected the body of data on the use of selected culture devices such as the rotating-wall vessel (57, 68). This review has two purposes not addressed by any of these other documents. The first is to summarize the engineering principles of suspension culture in a manner that will facilitate the choice of application-specific hardware, elucidate the limits of the technique, and aid in establishing optimal suspension culture conditions. Second, this review brings together available data on the mechanisms by which suspension culture mediates such dramatic changes in the properties of diverse types of cultured cells.
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PHYSIOLOGICAL IMPORTANCE |
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Although an old approach, suspension culture only increases in importance over time, as the demand for cell cultures maintaining differentiated features increases for both academic and industrial applications (24, 33, 68). Suspension culture continues to be useful for production of many bioproducts, from antibodies to hormones (31, 33, 36). The availability of cell culture models, which are so easily modulated and studied, with an intact tissue-specific repertoire of signal transduction and metabolic pathways would represent a dramatic biotechnology advancement. Hence, despite present limitations on the ability to access the basal cell membrane on beads for studies of transcellular transport, differentiated suspension culture cells have broad practical application. Engineering optimization of suspension culture was largely undertaken by National Aeronautics and Space Administration (NASA) engineers to model culture conditions in spaceflight (57, 72, 73) but may find its greatest utility in the carryover to ground-based applications (ftp://ftp.hq.nasa.gov/pub/pao/pressrel/2000/00-143.txt).
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TIMELINESS |
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The maturation of three major initiatives that utilize suspension culture techniques makes a review of the principles of suspension culture timely. First, an understanding of the physics and engineering principles is definitive but not freely available or reviewed in Medline-indexed journals (20, 22, 23, 36, 51, 52, 57, 72, 73). Many investigators contemplating suspension culture are likely to find a simple, direct summary extremely helpful.
Second, new methods to monitor and quantitate the effects of suspension culture on cells have become readily available (4, 16, 42, 43). The human genome project, and the associated production of huge libraries of expressed sequence tags, provided momentum for the development of new methods for assaying large numbers of genes simultaneously (4, 16, 42, 43). Gene (expression) array analysis has provided a rapid, inexpensive, but sophisticated method to meet these needs. Gene array has provided a revolution in the search for new drugs by monitoring a drug's effects on thousands of genes, making it possible to predict the usefulness of cell culture systems as model systems for both in vitro and in vivo toxicity testing (4, 16, 42, 43).
Finally, long used to produce antibodies and other products for clinical use, suspension cultures are now translating directly into clinical practice (33, 38, 56). Pancreatic islets are presently maintained alive, differentiated, and with regulated insulin secretion in rotating-wall vessel suspension culture before experimental and human transplantation (53-56, 59, 70). Suspension culture is used for industrial fermentation reactions (33), antibiotic production (38), interferon manufacture, and other bioproducts from hormones to hybridomas (35, 60; ftp://ftp.hq.nasa.gov/pub/pao/pressrel/2000/00-143.txt).
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OPTIMIZING CELL CULTURE IN SUSPENSION: THE ROTATING-WALL VESSEL |
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Engineering, Biology, and NASA
When biotechnology problems require an engineering solution, NASA's massive repository of engineering expertise has on occasion been applied to biological problems. Probably the best known and most fully developed example of this collaboration is the NASA/Baylor/DeBakey axial flow ventricular assist device or "artificial heart" (15, 31). After the heart failure death of a NASA engineer compromised by a prolonged wait for a donor transplant heart, NASA engineers played an integral role in the development of a low-shear, high-capacity blood pump. The NASA engineers initially donated thousands of man-hours and then continued with the support of a variety of Federal and private funding sources (15). NASA engineering was pivotal to the development of a pump combining the capacity to move the necessarily quite large blood volumes with preservation of the integrity of red blood cells and other blood elements (15, 31).Similarly, NASA's Biotechnology Group, based at the Johnson Space
Center in Houston, TX, has modeled the problem of optimizing mechanical
culture conditions in suspension culture by minimizing shear and
turbulence (22, 23, 51, 52, 57, 67, 72, 73). This led to a
radical new (but simple) design for suspension culture vessels embodied
in the rotating-wall vessel, a horizontally rotating cylindrical
culture vessel with a coaxial tubular oxygenator (Fig.
1). These vessels have characteristic
features that determine their utility. First, fluid flow is near solid
body or laminar at most operating conditions. This avoids the large
shear stresses associated with turbulent flow and allows introduction
of controlled and nearly homogenous shear fields. If the inner cylinder
and outer cylinders of the rotating-wall vessel rotate at the same angular velocity (rpm), then the laminar flow fluid velocity gradient (radially) is minimized. This minimizes the actual laminar flow and
totally eliminates the associated shear stress. The shear stress at any
point in the fluid is the product of the fluid velocity gradient and
viscosity. Second, the culture medium is gently mixed by rotation,
avoiding the necessity for stirring vanes. The mixing is the result of
a secondary flow pattern induced by particle sedimentation through the
fluid media or by laminar flows established when differential rotation
rates are chosen for the vessel components. Stirring vanes damage cells
by both local turbulence at their surface and the high flow rates
created between the vessel walls and the vanes. The vanes or any other
"in-vessel" mixing apparatus invariably causes inhomogeneity in the
shear field, often of large magnitudes, which confounds the
interpretation of culture results. Third, there is no headspace. In
roller bottles, due to incomplete filling of the vessel, the air in the
headspace creates turbulence and secondary bubble formation in the
culture medium, which are both potent sources of extra shear and
turbulence. Fourth, anchorage-dependent cell types can be grown on
microcarriers, just as in other suspension devices (23,
64). Last, the coaxial oxygenator and the outer wall may rotate
at the same slow (10-60 rpm) rate, avoiding and/or minimizing
shear from differential rotation (see below) or that produced by a vane
passing the stationary wall of a spinner flask.
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The rotating-wall vessel largely solves the challenges of suspension culture: to suspend cells and microcarriers without inducing turbulence, or large degrees of shear, while providing adequate nutrition and oxygenation. Earlier reviews of the most efficient bioreactor design and agitation regimens concluded that conditions of turbulent flow existed in all stirred tank bioreactors (7, 8, 12-14). The ability of cells to proliferate in horizontally rotating vessels had been demonstrated (3) but never optimized or combined with solutions of the oxygenation and nutrition problems. Oxygenation by bubbling or sparging is another potent source of mechanical cell impact and damage (6, 58). This problem is overcome by the continuous delivery of large volumes of oxygen along an axial oxygenator, using only dissolved gases without bubbles for exchange (22, 51, 72). Last, intermittent batch feeding or perfusion can provide nutrition (22, 51).
In summary, the rotating-wall vessel provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis that is characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion, excluding undissolved gases from the bioreactor (22, 51, 72, 73).
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PRINCIPLES OF OPTIMIZED SUSPENSION CULTURE IN THE ROTATING-WALL VESSEL: TRADEOFFS |
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The rotating-wall vessel has simple features designed to provide
continuous sedimentation of particles through culture medium, which is
rotating essentially as a solid mass with minimal induced cellular
shear and turbulence. Three tradeoffs inherent in the operation of the
rotating-wall vessel are key to both defining the limits of the
technology and optimizing the mechanical cell culture conditions
(19, 20, 34-36, 60, 72) (Table
1). The first tradeoff results from the
determinants of terminal velocity in the vessel. The second tradeoff
results from a balance of Coriolis and centrifugal forces induced by
rotation of the gravitational vector. Third, mass transfer is effected
by both terminal velocity and the diffusion properties of the culture
medium. To optimize suspension culture, these tradeoffs need to be
expressed in simple mathematical relationships, quantitated, and
controlled.
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Tradeoff I: Determinants of Terminal Velocity and Shear in the Rotating-Wall Vessel
Terminal velocity.
A cell or cell aggregate in suspension culture in the rotating-wall
vessel accelerates through the fluid until it reaches a terminal
velocity (Vs), at which the pull of gravity is
balanced by equal and opposite hydrodynamic forces including shear,
centrifugal, and Coriolis forces. The shear on a particle is
proportional to the terminal velocity of the cell aggregate, which is
in turn determined by Eq. 1 (22, 51, 67,
72)
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(1) |
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Shear stress.
The maximum shear stress (max) is a function of the
terminal velocity as shown by the following (1, 20, 72)
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(2) |
Practical implications. The physics of the rotating-wall vessel determines that the shear stress is fixed by gravity, and this has vital practical implications. This relationship means that every time the rotating-wall vessel containing the same cells and culture medium is turned on, it delivers exactly the same terminal velocity and consequent shear stress to similar-sized particles, no matter how much the rotation speed may vary. This is essentially a foolproof system. Contrast this with the physics of a spinner flask, in which minor changes in speed create dramatic changes in shear stress (22, 72) that are further confounding due to the dramatic time and spatial inhomogeneity of the shear field. Given the tiny dynamic range of shear stress in which many cultured cell types flourish (22, 57) before being irreversibly damaged by the ensuing physical forces, minor increases in spinner vane speed often have catastrophic consequences for the structural integrity of cell cultures.
Tradeoff II: Coriolis Spiraling Due to Radial and Tangential Components of the Gravity Vector
The resolution of the gravity vector acting on a particle in the rotating-wall vessel into radial and tangential components is of great practical importance as it explains and quantitates the slow spiraling of particles in the vessel and leads to an explanation of the limitations of the vessel's efficacy (and hence suspension culture efficacy) (20, 36, 46, 50, 51, 67, 72, 73).The circumference of the circular particle motion is only determined by the sedimentation rate and vessel rotation rate. Secondary, low-order Coriolis motions result only from the radial velocity components, which themselves are determined by gravity sedimentation and centrifugation. These are negligible in practice but contribute to a slow lagging of the particles behind the vessel's angular position.
As the fluid rotates in a rotating-wall vessel, the gravitational
vector acting on a particle can be resolved into radial and tangential
components (20, 36, 50, 72) (Fig.
2A). That is, the diameter of
the spirals of particles cultured in suspension in the rotating-wall
vessel is determined by the terminal velocity (see Eq. 1 and
Table 2), the angular velocity of the vessel, gravity, and the radial
particle velocity. These relationships may be more intuitively
expressed diagrammatically, where it can be seen that the amplitude of
the Coriolis spiraling induced by gravity varies exponentially with
vessel rotation speed in an inverse manner (Fig. 2B).
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Examples of Coriolis forces come in diverse sizes. One dramatic example of Coriolis forces in action is the spiraling of a hurricane. A hurricane is a unidirectional wind traveling across the spinning surface of the earth as it rotates. As the wind crosses the spinning globe, Coriolis forces induce the characteristic hurricane spiraling that is so familiar on weather maps (71).
The second tradeoff limiting the efficacy of the rotating-wall vessel occurs, in contrast to the exponential relationship between rpm and gravitational vectors, as centrifugal force is directly linearly proportional to rotation speed (Fig. 2B) (20, 37, 50, 72). Several practical applications of these relationships explain the behavior of particles in the rotating-wall vessel (72). At low rotation speeds, the gravity-induced deviations predominate and the spirals are extremely large, with many wall impacts. At its extension, at extremely low rotation speeds, as the rotations approach zero the spirals are so large that the particles make one nearly continuous wall impact, by sitting on the bottom of the vessel. At high rotation speeds, centrifugal forces dominant distortions, wall impacts again rise, and the particles stick to the outer wall of the vessel. These effects define the practical operating range for rotating-wall vessels. As larger particles, with correspondingly larger sedimentation rates, are cultured, we become less able to choose a rotational rate that minimizes wall impacts or contact.
Even more important than these boundary conditions are the limitations on particle size mediated by these gravity-dependent changes (20, 37, 50, 72). At low rotation speeds, increasing the rotation speed of the vessel (Fig. 2B, left, where gravitational effects are dominant) can minimize the deviations from an ideally circular course of the particles. As the particles increase in size, the terminal velocity increases, and more shear is required to keep the particles in suspension. Continued improvement due to reduction of gravitational effects as vessel rotation speed increases is offset and outstripped by increasing centrifugal effects (Fig. 2B, left to right, showing increasing rotation speed). As particles such as cell aggregates increase in size, high rpm is needed to keep them in suspension, and centrifugal forces become dominant in deviation from a perfectly circular rotation path. Wall impacts increase due to necessary increases in angular velocity, as well as an exponential increase in shear with increasing particle radius (Eq. 1). These limitations defined by the rotation required to suspend increasingly large particles in culture, combined with exponentially increasing shear with particle radius, define a limit to the unit gravity operation of the rotating-wall vessel for suspension culture. We observe significant effects as typical tissue aggregates approach 1 cm in diameter (72, 73).
The second tradeoff in the rotating-wall vessel is that rotation speed needs to be optimized by finding the angular velocity at which the total of Coriolis and centrifugal forces is minimized. Note carefully that both the Coriolis and centrifugal forces acting on particles in the vessel are directly related to the terminal velocity. If terminal velocity is minimized by changes in the mechanical culture conditions, the Coriolis and centrifugal forces are simultaneously minimized. However, only the gravity-induced motions reach steady state. The centrifugation and secondary, very small Coriolis effects are ever present, causing a slow drift outward and a phase lag with respect to the vessel wall. Within the operation range of the vessel, we usually do not observe the centrifugation outward or Coriolis effects because, in practice, other disturbances and fluid motions, such as motor vibrations, mask them.
To demonstrate the functioning of the vessel, Fig. 2C depicts the trajectory of a cell aggregate in the vessel using a stationary frame of reference. The aggregate spirals into a steady-state position in which the tradeoff of Coriolis and centrifugal forces is balanced. The actual particle trajectory during the turning of the rotating-wall vessel is shown in Fig. 2D. Hence, within a few rotations, particles come to a steady-state trajectory, in which the majority of the time they are close to the center of the fluid annulus of the vessel.
The interrelationships of several major determinants of terminal
velocity and shear in the rotating-wall vessel can be summarized in a
diagram (Fig. 3). Both terminal velocity
and shear are directly proportional to the difference in density
between particles in the vessel and the fluid media (Fig.
3A). Neither terminal velocity nor shear is affected at all
by rotation speed (Fig. 3B) (although rotation speed
determines the number of wall impacts). Particle radius has a direct
linear effect on terminal velocity and a more complex curvilinear
effect on shear (Fig. 3C). Media viscosity has a complex
curvilinear effect on terminal velocity but no effect on shear (Fig.
3D).
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Tradeoff III: Mass Transfer
The third tradeoff in the vessel is that changes in mechanical culture conditions also affect nutrient delivery to cells by convective bulk flow and diffusion. The delivery of nutrients to a cell in suspension culture depends on two factors: the "convective" motion of nutrients depends on the relative flow of nutrients and movement of the cell and on the diffusion rate of each solute in the particular culture medium (11, 19, 35, 60, 71).In conditions where there is no relative fluid motion (that is,
terminal velocity approaches 0), diffusion becomes the dominant mechanism for mass transport. The diffusional flux of a component in
the z-direction
(JA, z) is given as the
product of the diffusion coefficient of that species
(DAB) and the concentration gradient (11,
19, 71)
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The ratio of the convective motion to the diffusion rate is a dimensionless constant known as the Peclet number, which is highly predictive of mass transport limitations. For small particles, around the size of bacteria, with a Peclet number <10, diffusion can be limiting in suspension culture. For mammalian cells on beads with a Peclet number >10, mass transport tends to be convection, not diffusion, limited (11, 19, 35, 64, 71). In other words, a cell aggregate falling through the culture media in the rotating-wall vessel is fed as it effectively sieves through the nutrient media. It is only when the motion of cell aggregates and nutrients becomes equal, as terminal velocity approaches zero, that there is any role for diffusion in nutrition.
The practical application of the mass-transfer tradeoff is twofold. First, cell viability must be monitored in suspension culture to ensure that nutrient delivery has not become limiting. Second, this tradeoff suggests that it is better, when terminal velocity is reduced by driving particle and culture medium densities together, to utilize lighter microaggregate beads whenever possible rather then to increase the density of the culture media. Density increases also increase viscosity at the price of less diffusion, a metabolic cost avoided when the problem is solved by the use of lighter beads.
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VALIDATING THE MATHEMATICAL MODELS: OBSERVED VS. PREDICTED EFFECTS OF MECHANICAL CULTURE CONDITIONS ON PARTICLE MOTION |
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Importantly, the predicted particle pathways in rotating-wall vessel culture suggested by these relationships have been tested and validated experimentally (50, 73). NASA optimally engineered the low-shear, laminar flow conditions in the rotating-wall vessel in an attempt to develop a robust model system with many features of the low-shear, low-turbulence conditions predicted for cell culture in space. Hence many of the experimental validations of these principles have been based on changing gravitational conditions. Practical confirmation of the effects of gravity were obtained by video recording and quantitating the path of particles in a rotating-wall vessel during variations induced by the parabolic flight path of a NASA KC-135 aircraft. Popularly referred to as the "vomit comet," this aircraft has been utilized to provide repeated ~25-s periods of <0.1 gravity by following a looping flight path. The predicted and observed particle motion of beads at different forces in the rotating-wall vessel agreed to within <1%, and sources of error such as irregularities in the rotator drive motor speed or secondary fluid motions caused by particle motion were identified (72).
Robeck (50) wrote a computer simulation of this model and tested it against video data of cells growing in the rotating-wall vessel in microgravity on the space shuttle on space transportation system flight STS-70. There was excellent agreement between the predicted and observed particle paths in this experiment.
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OTHER WAYS OF DISRUPTING OPTIMAL CULTURE FLUID DYNAMICS |
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The optimized mechanical culture conditions can be disrupted in many other ways, such as differential rotation of inner and outer walls of the vessel or bubble formation. These disruptions are remarkable in that an apparently tiny change in fluid flow can have dramatically disproportionate effects on shear and turbulence (22).
Differential Rotation
To study the effects of reintroduction of shear in a structured quantifiable manner, it is desirable to be able to reinduce shear in the rotating-wall vessel. One simple modality to reinduce shear is to rotate the inner coaxial oxygenator (inner wall) and the outer wall at different speeds. This differential rotation has been used to reintroduce quantifiable levels of shear (22, 57). Counterrotation of inner and outer walls, with or without a mixing vane, can also be used to mix the contents of the vessel, but this incurs high amounts of shear. Differential rotation is potent in its ability to induce shear. This is where the real laminar flow gradient comes in and thus the specifically induced shear forces. In the case where the inner and outer angular velocities are equal, the fluid particles in the medium, once in equilibrium, should be rotating at essentially the same rotations per minute as the rotating-wall vessel itself. The major determinant of the amount of shear (
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Both theoretical and experimental approaches have validated the dynamic ranges of inner and outer wall rotation, together with associated shear stresses, which can be achieved before laminar flow degrades (1). Diverse forms of turbulence occur as laminar flow degrades, although the range of relative wall rotations at which laminar flow is preserved is predictable and has been simply summarized in both mathematical and diagrammatic formats (1).
Bubbles
The optimized engineering of the rotating-wall vessel in which shear and turbulence are minimized is entirely dependent on maintaining laminar flow in the culture vessel. These are several examples of simple perturbations, which induce turbulence and potently disrupt the advantages and biological efficacy of the vessel. The simplest and best modeled example is the effect of a single small bubble placed on the baseplate of the vessel (6, 36, 46). Such an apparently humble change induces turbulent eddies in the distal third of the vessel, with the exact pattern of turbulence heavily dependent on the size of the bubble (46). However, there are simple ways to prevent bubble formation by using suitably humidified dissolved gas mixtures. ![]() |
BEADS AND OTHER SCAFFOLDS |
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Many cells such as lymphocytes or bacteria can be grown in the rotating-wall vessel in suspension with no other support (10, 41, 57). Adhesion-dependent cells such as renal epithelial cells or prostate epithelial cells, which require an adherent base to proliferate, are commonly grown on beads (24, 25, 76). The most popular bead is a 175-µm-diameter collagen bead known as Cytodex-3 with specific gravity of 1.04. Beads manufactured of collagen and glass are commercially available in a variety of diameters and densities, allowing for simple modulation of the initial mechanical conditions. As all of these beads are difficult to impossible to section, structural studies other than scanning electron microscopy are often performed on Cultisphere gelatin beads, which are a porous honeycomb that is simple to section (24, 68). Similarly, some cells, such as cartilage, grow well on dissolvable polyglycolic acid matrices (17), which degrade completely over a matter of days in solution.
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CELLULAR AND MOLECULAR MECHANISMS OF ROTATING-WALL VESSEL ACTION |
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An understanding of the engineering principles of suspension culture is helpful, but for a cell biologist the molecular mechanisms by which the cellular changes are mediated are more interesting. There are precedents in the cell culture literature by which physical forces can induce changes in cell metabolism and function. Heat shock proteins, in which tiny changes in temperature induce a cascade of genetic and protein changes, are a well-documented example (5, 17, 74). More recently, there is abundant evidence than shear stress due to flow across the vascular endothelium is critical to maintaining many attributes of normal cell structure and function (21, 47, 48, 66).
The mechanisms by which mechanical culture conditions affect gene expression, protein content, and the structure and function of diverse cell lines is only beginning to be examined systematically. The simplest explanation of the transduction of gravity-dependent effects occurs in cells with sedimenting organelles such as otic hair cells or statoliths in plants. The altered mechanical culture conditions of spaceflight have dramatic effects on cell growth and metabolism, as well as the production of bioproducts from growth hormone to hybridomas (reviewed in Refs. 33, 60 and 61). Postulated mechanisms include cytoskeletal elasticity changes, electrical and chemical signaling, and channel activation (60-65), although there has been no systematic experimental dissection of these mechanisms to date. There are, however, a few experimental clues as to mechanism, including a role for shear stress (32), gene array investigation of patterns of genetic response (24, 25, 32), definition of some signal transduction changes (9, 37), and modeling of the effects of the vessel on both the cell cycle (28), and pathways of cell death (11).
Shear Stress
Fluid shear stresses generated by blood flow in the vasculature can profoundly influence the phenotype of the endothelium by regulating the activity of certain flow-sensitive proteins (enzymes, for example) as well as by modulating gene expression (21, 47, 48, 66). The finding that specific fluid mechanical forces can alter endothelial structure and function has provided a framework for a mechanistic understanding of flow-dependent processes, ranging from vascular remodeling in response to hemodynamic changes to the initiation and localization of chronic vascular diseases such as atherosclerosis (21, 66).The effects of shear stress on vascular endothelial cells raised obvious questions about similar responses in other cell types. Our laboratory provided evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells (32). Rotating-wall culture of renal cells changes expression of select gene products, including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM, and MnSOD. With the use of a putative endothelial cell shear stress response element binding site as a decoy, the role of this sequence in the regulation of selected genes in epithelial cells was demonstrated (32).
Gene Expression
Several lines of evidence suggest that physical factors can change cellular gene expression, but no one had addressed the extent or nature of these changes quantitatively (24, 25). To elucidate the specific mechanical parameters responsible for the genetic and morphological changes, we grew primary human renal cell cultures under conditions providing differences in three-dimensionality, cospatial relationship of cells, and shear (24, 25) (Table 3). Each culture derived from the same stock of human renal primary cells was grown for 6 days. The rotating-wall vessel was the best ground-based optimization of the mechanical parameters. To fully optimize these mechanical parameters, aliquots of the cells were grown in space on the space shuttle, with fixation on day 6. Culture of the cells during exposure to a force of three times the earth's gravity, achieved by placing culture bags in a centrifuge, was used as a negative control as none of the mechanical conditions hypothesized to be relevant were modulated. Gene expression analysis using microarray technology allows detailed observations of genetic expression under various culture conditions, with quantitative comparison of the nature, grouping, and extent of changes.
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More than 1,632 genes changed more than twofold at 6 days during
microgravity culture on space transportation system flight STS-90
"Neurolab." These changes were not known shear stress response elements and heat shock proteins (shown in green in Fig.
4). Specific transcription factors (shown
in red), including the Wilms tumor zinc finger protein and the vitamin
D receptor, underwent large changes in microgravity (full data set at
web site http://ww.tmc.tulane.edu/astrobiology/microarrays). In the
rotating-wall vessel, 914 genes changed expression relative to the bag
control. Those genes that changed most on the microgravity array are
randomly spread throughout the rotating-wall vessel array,
demonstrating that the changes in gene expression observed in
microgravity are unique and not just an extension of the rotating-wall vessel. Only five genes changed more than threefold during
3-g centrifugation (24, 25).
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An escape from the tradeoff of balancing forces by examining the gravity-limited phenomenon of suspension culture in true microgravity demonstrated a dramatic degree of change in gene expression. Microgravity studies suggest unique mechanisms of tissue differentiation, which are distinct from the present best ground-based simulations (24, 25).
Signal Transduction
There are select phenomena in which specific signal transduction elements active during rotating-wall vessel culture have been defined. Activation of purified T cells with cross-linked CD2/CD28 and CD3/CD28 antibody pairs were completely suppressed in the rotating-wall vessel, suggesting a defect in signal transduction (10). Activation of purified T cell protein kinase C (PKC) with phorbol 12-myristate 13-acetate or calcium-dependent ionomycin activation were unaffected by rotating-wall vessel culture. Furthermore, submitogenic PKC-dependent doses of phorbol 12-myristate 13-acetate alone but not ionomycin alone restored phytohemoagglutinin responsiveness of peripheral blood mononuclear cells in rotating-wall vessel culture. These data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to mechanical culture conditions.PC12 cells cultured under "simulated microgravity" conditions showed altered patterns of protein tyrosine phosphorylation and prolonged activation of c-fos, a member of the AP-1 nuclear transcription factor complex (39).
Apoptotic/Necrotic Pathway Monitoring
To more fully understand the effects that rotating-wall vessel cultivation has on cellular longevity, the mode and kinetics of Spodoptera frugiperda cell death was studied and modeled in the quiescent environment of the rotating-wall vessel and compared with a shaker-flask control (11). Data from flow cytometry and fluorescence microscopy show a twofold greater accumulation of apoptotic cells in the rotating-wall vessel culture (Fig. 5). Kinetic constants reveal that total cell death is reduced in the rotating-wall vessel, and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found to be independent of culture conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first-order term. Cell-cycle analysis shows that the percentage of tetraploid cells gradually decreases during cultivation in both vessels. This suggests the tetraploid phase as the likely origin for apoptosis in these cultures. These studies provide a means for further enhancing culture longevity.
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These findings in the rotating-wall vessel are consistent with other model systems (75). When six hybridoma cell lines grown under diverse experimental conditions in both normal continuous and perfusion cultures are analyzed with respect to the significance of nutrients and products in determining the growth and death rates, the specific growth rate (µ) almost linearly correlates with the ratio of the viable cell concentration (NV) to the dilution (perfusion) rate (D). Similarly, the specific death rate (kd) is a function of the ratio of the total cell concentration (Nt) to the dilution (perfusion) rate, which strongly suggests the formation of not yet identified critical factors or autoinhibitors that determine both the growth and death rates of hybridoma cells. That is, the cells produce a bioproduct that accumulates in the culture medium and effects changes in cell cycle and cell death in a concentration-dependent manner.
Role of the Cytoskeleton
The central role of the cytoskeleton in cell scaffolding, load bearing, and transportation of vesicles, continues to make it a popular candidate for transducing physical stimuli into other cellular processes (40, 41, 64). There is some molecular evidence for this process, including concomitant cytoskeletal alterations characterized by diffuse, shortened microtubules, increased apoptosis, and time-dependent elevation in Fas/APO-1 protein in space-flown human lymphocytes (40, 41). ![]() |
OTHER BENEFITS OF OPTIMIZED ROTATING-WALL VESSEL CULTURE: COCULTURE, NUTRIENT DELIVERY, AND EASE OF REAGENT EXPOSURE |
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The rotating-wall vessel provides some other advantages relevant to many forms of tissue culture. The vessel supports coculture efficiently by bringing different cell types of different size and density together simply and efficiently. It is very simple to expose the suspension cultures to reagents in the vessel. These properties of the vessel are especially important for tissue engineering studies, one of the areas in which utilization of the rotating-wall vessel has been most active. The rotating-wall vessel has been successful in engineering prostate organoids (49, 76), colon carcinoma (28), and cartilage (18, 69), among other tissues (68).
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NOMENCLATURE: SIMULATED MICROGRAVITY VS. OPTIMIZED SUSPENSION CULTURE |
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As the rotating-wall vessel was developed by NASA to simulate, as far as possible, culture conditions predicted to occur during experiments in space, these conditions were given the moniker simulated microgravity (22, 72). Many articles have been published as "the effects on simulated microgravity on... ." Without one's knowledge of the purpose and history of this term, it tends to be confusing, as it suggests the physical impossibility that there is no gravity inside the vessel. These reviewers strongly advocate a return to descriptive, intuitively informative nomenclature: the rotating-wall vessel changes mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence, not simulated microgravity.
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CONCLUSIONS |
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To minimize mechanical damage to cultured cells, suspension cultured should be performed under conditions of near-solid body or laminar flow. This is most simply achieved in a solid-body rotation Couette-flow, zero-head-space culture vessel such as the rotating-wall vessel. The rotating-wall vessel provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis that is characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. The utility of turbulent flow culture devices such as roller bottles or stirred fermentors is limited by the severe mechanical stresses they induce.
Optimization of suspension culture is achieved by application of three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Reducing or removing gravity, which likely underlies many of the effects of spaceflight on cell culture, can also minimize terminal velocity.
Second, rotation in the rotating-wall vessel induces Coriolis and centrifugal forces. Both of these forces are directly dependent on terminal velocity and are minimized as terminal velocity is minimized.
Third, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. As terminal velocity is minimized, nutrient delivery by mass flow is also reduced. Diffusion depends on the properties of both the individual nutrient and the culture medium. For small particles such as bacteria, diffusion can limit nutrient supply in suspension culture, but for large particles such as mammalian cells on microcarrier beads mass transit is likely to be terminal velocity limited. Both these limitations to nutrient supply can be overcome by lowering the viscosity of the culture medium and/or providing a nutrient supply to form a concentration gradient. Introduction of differential vessel component rotation rates or incorporation of devices such as the viscous pump (36) allows induction of arbitrary levels of active mixing with highly controlled and homogenous fluid mechanical conditions. This is particularly important in spaceflight, where particle sedimentation is absent.
Several lines of evidence support a role for multiple molecular mechanisms in the transduction of mechanical culture conditions into cellular effects. These mechanisms include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as PKC. New methods of gene array analysis are beginning to dissect these mechanistic possibilities.
Finally, the discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and biological effects, with an emphasis on cellular processes important for the industrial production of biological pharmaceuticals and devices.
The application of suspension culture has been limited by the failure to develop a simple optimization strategy based on mechanical culture conditions. This review may provide a roadmap to facilitate both industrial and academic applications to help fulfill the massive promise of suspension culture.
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ACKNOWLEDGEMENTS |
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This work was supported by NASA NRAs Basic 9-811, NAG-8-1362, and Co-operative agreement NCC 2-1177. The rotating-wall vessel is manufactured under license from NASA by Synthecon of Houston, TX (see www.synthecon.com). Screaming Jeanne's, Inc., is a genetic diagnosis and cell culture company in New Orleans, LA.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. G. Hammond, Nephrology Section SL45, Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: thammond{at}tulane.edu).
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