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Shear sensitivity in animal cell culture
Shear sensitivity in animal cell culture Keith J. Gooch and John A. Frangos Pennsylvania State University, University Park, Pennsylvania, USA Over the past year, considerable progress has been made in understanding shear sensitivity in animal cell culture as a result of extensive theoretical and experimental work. Here we review this progress, paying special attention to the physical and biological mechanisms by which mechanical forces act upon cells, and the effects of such forces. Current Opinion in Biotechnology 1993, 4:193-196
Introduction All cells are constantly exposed to mechanical forces in vivo. In the technical literature, these forces are considered primarily as destructive elements, resulting in cell death. However, they may also modify the morphology, increase the growth rate, and alter the metabolic activity of cells. Even though animal cells are often more sensitive than prokaryotic cells to mechanical forces as a result of their relatively large size and lack of cell wall, they are used in many important cell-culture applications such as the production of antibodies or proteins requiring post-translational modifications, experimental cancer therapies, and biological and medical research.
Origin of mechanical forces To understand shear sensitivity in animal cell culture, it is necessary to understand the origin and magnitude of mechanical forces, as well as the effects these forces have on cells. Most current research on the origin and magnitude of mechanical forces has attempted to correlate these forces with cell death. It is important, however, to remember that cell death is only one of many significant cellular responses. Mechanical forces that act u p o n a cell arise as a result of interactions between the cell and its surroundings. In all cell-culture systems, the bulk fluid exerts a hydrostatic pressure on the cell. If the fluid is in motion, shear forces and additional pressure forces occur. In cultures of freely suspended or anchorage-dependent cells grown on microcarrier beads, fluid motion also results in interactions between cells or their carriers with other cells, microcarrier beads, or other solid surfaces such as the impellers or vessel walls. The presence of a gas phase introduces additional forces as a result of bubble formation, translation, and rupture.
have attempted to correlate the frequency and/or intensity of particle-to-particle or particle-to-eddy interactions with cell death. In these models, both freely suspended cells and microcarrier beads may be considered as a particle. Experimental evidence indicates that partide-to-particle interactions are the main cause of damage to cells grown on microcarriers under conditions of intense agitation [1"]. Most models of particle-to-eddy interactions emphasize interactions between particles and eddies of a similar size. The reason for this is that interactions between a particle and a larger eddy merely result in the displacement of the particle without it absorbing a significant amount of energy. However, interactions between a particle and an eddy of similar size may result in the energy of the eddy being transferred to the particle [1"]. A new theoretical model that accounts for both particle-to-particle and particle-to-eddy interactions has recently been developed [21. This model predicts that the rate of cell death resulting from agitation should decrease with increasing medium viscosity and that the protective effect of increased viscosity is more significant at greater agitation intensities. The predictions of this theoretical model were tested experimentally and found to be correct [21. In bioreactors where bubbles are present as a result of sparging or entrainment of gas from the surface interface, they are often the most significant cause of cell death. Microscopic visualization has shown that animal cells may adsorb to a bubble as it rises through the bulk fluid, especially as it passes through the liquid-gas interface at the liquid surface [3,4]. Theoretical predictions of the forces on a cell in a bursting bubble indicate that these forces are sufficient to cause death of the cell [51. Cherry and HuUe [5] developed the following theoretical equation for the first-order death rate constant in an air lift reactor, as the result of bubble translation and rupture:
Most models of injury occurring to cells caused by agitation in the absence of entrained or sparged bubbles,
© Current Biology Ltd ISSN 0958-1669
t.3hF,
193
194
Biochemicalengineering This equation predicts that k is proportional to the volumetric flow rate of gas through the sparger, F, and inversely proportional to both the bubble radius, r, and the reactor volume, V. The parameters ~ (the fraction of ceils present in a bubble that are killed w h e n the bubble bursts) and Cf/Cb (the concentration of cells present in the bubble film divided b y the concentration of cells in the bulk liquid) were measured in independent experiments and were 0.2 and 0.6 respectively for the system used. If h, the thickness of the bubble film immediately before bursting, is assumed to b e equal to the average cell diameter, the calculated value for k is similar to that observed experimentally for their air-lift reactor. It is s o m e w h a t surprising that Cf/CB is less than unity as bacteria, algae, and protozoa are thought to concentrate at liquid-gas interfaces. This anomaly may be the result of differences in m e m b r a n e hydrophobicity b e t w e e n the various cell types.
The mechanical properties of cells To understand the effects of fluid-mechanical forces on a cell, knowledge of the mechanical properties of the cell is required. By applying k n o w n forces to an individual cell and observing its deformation and/or lysis, it is possible to measure the mechanical properties of the cell. One method of determining cortical tension and apparent viscosity of the cell is to aspirate the cell into a micropipette with a diameter less than that of the cell, and observe its rate of deformation. This method was utilized b y Needham et al. [6] to characterize a hybridoma cell line. They found that the mechanical properties of hybridomas, unlike erythrocytes, cannot be characterized b y a single value for a given parameter, but instead exhibit a wide range of values that are d e p e n d e n t on the growth stage of the cell. Another m e t h o d of measuring a cell's physical properties is to compress it b e t w e e n two surfaces and measure the force required for deformation and lysis. Utilizing this method, Zhang et al. [7,8] s h o w e d that the bursting strength of a cell is dependent on its size, while the m e a n compressibility modules, bursting m e m b r a n e tension, and relative increase of cell area at bursting, all vary with the age of the culture but are independent of cell size.
Methods of reducing the effects of mechanical forces on cells It is often desirable to reduce cell shear sensitivity. This may be accomplished either by decreasing the intensity (frequency, duration, rate of change and/or magnitude) of the physical forces acting on the cells or by increasing the resistance of the cells to physical forces. In practice, both of these objectives may be achieved b y the use of media additives such as natural or synthetic polymers or serum. The mechanisms by which
additives decrease shear sensitivity are not well understood but may include altering plasma m e m b r a n e fluidity [9q or bulk fluid properties such as viscosity [2], or by excluding cells from potentially high shear regions such as liquid-gas interfaces [10]. The use of additives to reduce shear sensitivity of cells has recently b e e n reviewed b y Goosen [11] and by Papoutskis [12"] so will not b e discussed extensively here. Shear sensitivity may be altered as a result of cell culture conditions. A recent study of deformation-dependent potassium leakage in red blood cells revealed that physiological levels of peroxide d a m a g e induced by the addition of t-butylhydroperoxide dramatically increased sensitivity to non-lytic shear [13]. It is possible that s o m e additives m a y work by reducing biological or chemical stresses on a cell, thus making it more resistant to mechanical forces. Another m e t h o d of reducing shear-induced effects is to modify bioreactor design or operating conditions. G o o s e n [11] discusses several recent advances in lowshear continuous bioreactors, including the use of a helical ribbon impeller and the d e v e l o p m e n t of an air-lift/fiber-bed bioreactor. The use of a tubular microporous m e m b r a n e aerator can eliminate the presence of bubbles and their associated forces. A theoretically optimal design of such an aerator has b e e n p r o p o s e d recently [14]. A novel technique for reducing shear sensitivity is the use of perfluorocarbon emulsions. Perfluorocarbon decreases the n e e d for sparging and agitation b y increasing oxygen transfer and medium density (thereby decreasing the tendency of cells to settle). In addition, these emulsions protect cells from d a m a g e resulting from aeration, possibly b y forming a stable foam layer at the surface of the bioreactor which the cells cannot penetrate [10].
The effects of mechanical forces on cells It has long b e e n established that endothelial cells are able to modify their morphology, growth rate, and metabolism in response to changes in fluid flow. Recent w o r k has b e g u n to elucidate the mechanisms b y which endothelial cells respond to flow. Flow-induced production of platelet-derived growth factor mRNA and prostacyclin appears to follow similar signal transduction pathways. Fluid flow results in activation of G proteins that in turn activate phospholipases [15,16"]. Phospholipases then cleave phospholipids, including phosphatidylinositol, phosphatidylyethanolamine, and phosphatidic acid [17]. It appears that the products of the phospholipid cleavage then act as secondary messengers that activate the cell. The response of the cell to the secondary messenger is not a non-specific activation, but a specific and coordinated metabolic change. For example, flow induces endothelial cells to produce endothelium-derived relaxing factor, a vasodila-
Shear sensitivity in animal cell culture Gooch and Frangos tor, while subsequently inhibiting the production of endothelin-1, a powerful vasoconstrictor [18]. The components of the signal transduction pathway, such as G proteins and phospholipases, are found not only in endothelial cells, but in all animal cells. Therefore, it is not surprising that other cell types such as osteoblasts and fibroblasts [19], and kidney tubule cells [20] are also stimulated by flow. Recent w o r k with Chinese hamster ovary cells cultured in suspension in a two liter bioreactor shows that these cells are also stimulated by mechanical forces [21"]. Although increased agitation intensity decreases cell viability, increased agitation induces the viable cells to proliferate more rapidly c o m p a r e d to low agitation control cultures. The increased proliferation rate was demonstrated b y flow cytometry, which revealed an increase in the rate of DNA synthesis and an increased fraction of viable cells in S phase. The proliferation rate remained elevated for at least six hours after the agitation rate was returned to control levels as demonstrated by a continued elevated rate of DNA synthesis and intrinsic cellular growth.
Conclusion Animal cells in culture are constantly exposed to mechanical forces. Excessive forces can cause cell death, though several practical and effective methods of protecting cells do exist. Smaller forces can produce more subtle, but important, changes in morphology, growth rate, and metabolic activity.
Acknowledgements This work was supported by the United States National Heart, Lung a n d Blood Institute Grant HL40696. JA Frangos is a recipient of the National Science Foundation Presidential Young Investigator Award. KJ G o o c h is a recipient of a National Institute of Health Biotechnology Training Fellowship.
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1.
PAPOLrrSAKIS E: F l u i d - M e c h a n i c a l D a m a g e o f A n i m a l Cells i n B i o r e a c t o r s . Trends Biotechnol 1991, 9:427-437. A review of theoretical models a n d experimental data of fluid mechanical d a m a g e as a result of particle-to-eddy, particle-to-particle, a n d cell-to-bubble interactions. For freely s u s p e n d e d cells grown in Ilfixed bioreactors, most cell damage is caused by bubble breakup or fast-draining liquid films around bubbles, while in microcarrier bioreactors d a m a g e is caused by interactions between microcarriers with other microcarriers and also with small turbulent eddies. 2.
LAKHOTIAS, pAPOUTSAKISE: Agitation Induced Cell I n j u r y i n M i c r o c a r r i e r c u l t u r e s . P r o t e c t i v e Effect o f Viscos-
ity is Agitation Intensity Dependent: Experiments and M o d e l i n g . Btotechnol Btoeng 1992, 39:95-107. 3.
BAVARIANF, FAN L, CHALME~ J: M i c r o s c o p i c VislmliTation of Insect Cell-Bubble Interactions. h RisIng Bubbles, A i r - M e d i u m Interface, and the Foam Layer. Biotechnol Prog 1991, 7:140-150.
4.
CHALMERSJ, BAVARIANF: M i c r o s c o p i c Visu~liTation o f Ins e c t Cell-Bubble I n t e r a c t i o n s . II: T h e B u b b l e F i l m and Bubble Rupture. Biotechnol Prog 1991, 7:151-158.
5.
CHERRYR, HULLE C: Cell Death in the Thin Films o f B u r s t i n g B u b b l e s . Biotechnol Prog 1992, 8:11-18.
6.
NEEDHAMD, TING-BEALL H, TRAN-SON-TAY R: A Physical C h a r a c t e r i z a t i o n o f GAP A3 H y b r i d o m a Cells: Morphology, Geometry, a n d M e c h a n i c a l P r o p e r t i e s . Biotechnol Bioeng 1991, 38:8384352.
7.
ZHANGZ, FERENCZI M, LUSH A, THOMAS C: A N o v e l Mic r o m a n i p u l a t i o n T e c h n i q u e f o r M e a s u r i n g t h e Bursti n g S t r e n g t h o f SIngle Mamm21i~*a Cells. Appl Microbiol Btotechnol 1991, 36:208-210.
8.
ZHANGZ, AL-RUBEAIM, THOMAS C: M e c h a n i c a l P r o p e r t i e s o f I i y b r i d o m a Cells i n B a t c h C u l t u r e . Bfotechnol Left 1992, 14:11-16.
RAMIREZO, MLrrHARASAN R: Effect o f S e r u m o n the P l a s m a M e m b r a n e Fluidity o f Hybridomas: an I n s i g h t into its Shear Protective Mecltanlsm. Biotechnol Prog 1992, 8:40-50. By measuring steady-state fluorescence anisotropy, it was determ i n e d that increasing s e r u m concentration, which is k n o w n to decrease shear sensitivity, decreased plasma m e m b r a n e fluidity. Cholesterol enrichment of the plasma m e m b r a n e also decreased plasma m e m b r a n e fluidity a n d shear sensitivity. 9.
10.
Jo L, ARMIGER W: Use o f P e r ' f l u o r o c a r b o n E m u l s i o n s i n Cell c u l t u r e . Biotechniques 1992, 12:258-263.
11.
GOOSEN M: Large-Scale I n s e c t Cell C u l t u r e . Curr Opin Biotechnol 1992, 3:99-104.
PAPOUTSAKISE: M e d i a Additives for P r o t e c t i n g F r e e l y Suspended Animal Cells A g a i n s t A g i t a t i o n and Aeration. Trends Btotechnol 1991, 9:316-324. A review of the use of media additives to decrease shear sensitivity. General m e t h o d s for evaluating the effectiveness of a given additive as well as the effectiveness of s o m e c o m m o n l y u s e d additives are discussed. 12.
13.
HEBBEL R, MOHANDAS N: Reversible DeformationDependent Erythrocyte Cation Leak Extreme Sensitivity Conferred b y Minimal Peroxidation. Btophys J 1991, 60:712-715.
14.
Su W, CARAM H, HUMPHREY A: O p t i m a l D e s i g n o f the T u b u l a r M i c r o p o r o u s Membrane Aerator for ShearSensitive Cell C u l t u r e s . Biotechnol Prog 1992, 8:19-24.
15.
HSIEHH, LI N, FRANGOSJ: Shear-Induced P l a t e l e t - D e r i v e d Growth Factor Geue Expression In Human Endothelial Cells is M e d i a t e d b y P r o t e i n Kinase C. J Cell Physiol 1992, 150:552-558.
BERTHIAUMEF, FRANGOSJ: Flow-Induced P r o s t a c y c l i n Prod u c t i o n is Mediated by a P e r t u s s i s T o x i n - S e n s i t i v e G P r o t e i n . FEBS Lett 1992, 308:277-279. This paper presents data showing that fluid flow, like several hormonal agonists, induces prostacyclin production via a G protein d e p e n d e n t pathway. Flow potentiated the h o r m o n e - i n d u c e d production of prostacydin. 16.
17.
BHAGYALAKSHMIA, BERTHIAUME F, REICH M, FRANGOS J: F l u i d Shear Stress Stimmlates Membrane Phospholipid M e t a b o l i s m i n C u l t u r e d H u m a n E n d o t h e l i a l Cells. J Vasc Res 1992, 29:443-449.
18.
KUCHAN M, FRANGOS J; Shear Stress R e g u l a t e s E n d o t h e l i n - 1 R e l e a s e Via P r o t e i n K i n a s e C a n d cGMP
195
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Biochemical engineering in Cultured Endothelial 264:H150-H156.
Cells.
Am J Pbysiol 1993,
19.
REICH K, GAY C, FRANGOSJ: F l u i d S h e a r S t r e s s a s a Med i a t o r o f O s t e o b l a s t Cyclic A d e n o s i n e M o n o p h o s p h a t e P r o d u c t i o n . J Cell Phystol 1990, 143:100-104.
20.
KAWAHARAK, MATSUZAKIK: A c t i v a t i o n o f C a l c i u t n C h a n n e l b y S h e a r i n C t d t u r e d R e n a l Distal T u b u l e c e l l s . Biochem Biophys Res Commun 1992, 184:198-205.
21. •.
LAKHOTIA S, BAUER K, PAPOUTSAKIS E: D a m a g i n g &gitat i o n I n t e n s i t i e s I n c r e a s e DNA S y n t h e s i s Rate a n d A l t e r
c e l l - C y c l e P h a s e D i s t r i b u t i o n s o f CHO Cells. Bfotechnol Btoeng 1992, 40:978-990. A two-color flow cytometry m e t h o d was u s e d to determine the relative rate of DNA synthesis a n d t h e distribution of cells throughout the cell cycle. Increased agitation intensity decreases celt viability while simultaneously stimulating cells to proliferate more rapidly. This increased rate of proliferation lasted for at least six hours, even if agitation was returned to basal levels.
KJ Gooch a n d J A Frangos, 150 Fenske Laboratory, The Pennsylvania State University, University Park, PA 16802-4400, USA.
Introduction All cells are constantly exposed to mechanical forces in vivo. In the technical literature, these forces are considered primarily as destructive elements, resulting in cell death. However, they may also modify the morphology, increase the growth rate, and alter the metabolic activity of cells. Even though animal cells are often more sensitive than prokaryotic cells to mechanical forces as a result of their relatively large size and lack of cell wall, they are used in many important cell-culture applications such as the production of antibodies or proteins requiring post-translational modifications, experimental cancer therapies, and biological and medical research.
Origin of mechanical forces To understand shear sensitivity in animal cell culture, it is necessary to understand the origin and magnitude of mechanical forces, as well as the effects these forces have on cells. Most current research on the origin and magnitude of mechanical forces has attempted to correlate these forces with cell death. It is important, however, to remember that cell death is only one of many significant cellular responses. Mechanical forces that act u p o n a cell arise as a result of interactions between the cell and its surroundings. In all cell-culture systems, the bulk fluid exerts a hydrostatic pressure on the cell. If the fluid is in motion, shear forces and additional pressure forces occur. In cultures of freely suspended or anchorage-dependent cells grown on microcarrier beads, fluid motion also results in interactions between cells or their carriers with other cells, microcarrier beads, or other solid surfaces such as the impellers or vessel walls. The presence of a gas phase introduces additional forces as a result of bubble formation, translation, and rupture.
have attempted to correlate the frequency and/or intensity of particle-to-particle or particle-to-eddy interactions with cell death. In these models, both freely suspended cells and microcarrier beads may be considered as a particle. Experimental evidence indicates that partide-to-particle interactions are the main cause of damage to cells grown on microcarriers under conditions of intense agitation [1"]. Most models of particle-to-eddy interactions emphasize interactions between particles and eddies of a similar size. The reason for this is that interactions between a particle and a larger eddy merely result in the displacement of the particle without it absorbing a significant amount of energy. However, interactions between a particle and an eddy of similar size may result in the energy of the eddy being transferred to the particle [1"]. A new theoretical model that accounts for both particle-to-particle and particle-to-eddy interactions has recently been developed [21. This model predicts that the rate of cell death resulting from agitation should decrease with increasing medium viscosity and that the protective effect of increased viscosity is more significant at greater agitation intensities. The predictions of this theoretical model were tested experimentally and found to be correct [21. In bioreactors where bubbles are present as a result of sparging or entrainment of gas from the surface interface, they are often the most significant cause of cell death. Microscopic visualization has shown that animal cells may adsorb to a bubble as it rises through the bulk fluid, especially as it passes through the liquid-gas interface at the liquid surface [3,4]. Theoretical predictions of the forces on a cell in a bursting bubble indicate that these forces are sufficient to cause death of the cell [51. Cherry and HuUe [5] developed the following theoretical equation for the first-order death rate constant in an air lift reactor, as the result of bubble translation and rupture:
Most models of injury occurring to cells caused by agitation in the absence of entrained or sparged bubbles,
© Current Biology Ltd ISSN 0958-1669
t.3hF,
193
194
Biochemicalengineering This equation predicts that k is proportional to the volumetric flow rate of gas through the sparger, F, and inversely proportional to both the bubble radius, r, and the reactor volume, V. The parameters ~ (the fraction of ceils present in a bubble that are killed w h e n the bubble bursts) and Cf/Cb (the concentration of cells present in the bubble film divided b y the concentration of cells in the bulk liquid) were measured in independent experiments and were 0.2 and 0.6 respectively for the system used. If h, the thickness of the bubble film immediately before bursting, is assumed to b e equal to the average cell diameter, the calculated value for k is similar to that observed experimentally for their air-lift reactor. It is s o m e w h a t surprising that Cf/CB is less than unity as bacteria, algae, and protozoa are thought to concentrate at liquid-gas interfaces. This anomaly may be the result of differences in m e m b r a n e hydrophobicity b e t w e e n the various cell types.
The mechanical properties of cells To understand the effects of fluid-mechanical forces on a cell, knowledge of the mechanical properties of the cell is required. By applying k n o w n forces to an individual cell and observing its deformation and/or lysis, it is possible to measure the mechanical properties of the cell. One method of determining cortical tension and apparent viscosity of the cell is to aspirate the cell into a micropipette with a diameter less than that of the cell, and observe its rate of deformation. This method was utilized b y Needham et al. [6] to characterize a hybridoma cell line. They found that the mechanical properties of hybridomas, unlike erythrocytes, cannot be characterized b y a single value for a given parameter, but instead exhibit a wide range of values that are d e p e n d e n t on the growth stage of the cell. Another m e t h o d of measuring a cell's physical properties is to compress it b e t w e e n two surfaces and measure the force required for deformation and lysis. Utilizing this method, Zhang et al. [7,8] s h o w e d that the bursting strength of a cell is dependent on its size, while the m e a n compressibility modules, bursting m e m b r a n e tension, and relative increase of cell area at bursting, all vary with the age of the culture but are independent of cell size.
Methods of reducing the effects of mechanical forces on cells It is often desirable to reduce cell shear sensitivity. This may be accomplished either by decreasing the intensity (frequency, duration, rate of change and/or magnitude) of the physical forces acting on the cells or by increasing the resistance of the cells to physical forces. In practice, both of these objectives may be achieved b y the use of media additives such as natural or synthetic polymers or serum. The mechanisms by which
additives decrease shear sensitivity are not well understood but may include altering plasma m e m b r a n e fluidity [9q or bulk fluid properties such as viscosity [2], or by excluding cells from potentially high shear regions such as liquid-gas interfaces [10]. The use of additives to reduce shear sensitivity of cells has recently b e e n reviewed b y Goosen [11] and by Papoutskis [12"] so will not b e discussed extensively here. Shear sensitivity may be altered as a result of cell culture conditions. A recent study of deformation-dependent potassium leakage in red blood cells revealed that physiological levels of peroxide d a m a g e induced by the addition of t-butylhydroperoxide dramatically increased sensitivity to non-lytic shear [13]. It is possible that s o m e additives m a y work by reducing biological or chemical stresses on a cell, thus making it more resistant to mechanical forces. Another m e t h o d of reducing shear-induced effects is to modify bioreactor design or operating conditions. G o o s e n [11] discusses several recent advances in lowshear continuous bioreactors, including the use of a helical ribbon impeller and the d e v e l o p m e n t of an air-lift/fiber-bed bioreactor. The use of a tubular microporous m e m b r a n e aerator can eliminate the presence of bubbles and their associated forces. A theoretically optimal design of such an aerator has b e e n p r o p o s e d recently [14]. A novel technique for reducing shear sensitivity is the use of perfluorocarbon emulsions. Perfluorocarbon decreases the n e e d for sparging and agitation b y increasing oxygen transfer and medium density (thereby decreasing the tendency of cells to settle). In addition, these emulsions protect cells from d a m a g e resulting from aeration, possibly b y forming a stable foam layer at the surface of the bioreactor which the cells cannot penetrate [10].
The effects of mechanical forces on cells It has long b e e n established that endothelial cells are able to modify their morphology, growth rate, and metabolism in response to changes in fluid flow. Recent w o r k has b e g u n to elucidate the mechanisms b y which endothelial cells respond to flow. Flow-induced production of platelet-derived growth factor mRNA and prostacyclin appears to follow similar signal transduction pathways. Fluid flow results in activation of G proteins that in turn activate phospholipases [15,16"]. Phospholipases then cleave phospholipids, including phosphatidylinositol, phosphatidylyethanolamine, and phosphatidic acid [17]. It appears that the products of the phospholipid cleavage then act as secondary messengers that activate the cell. The response of the cell to the secondary messenger is not a non-specific activation, but a specific and coordinated metabolic change. For example, flow induces endothelial cells to produce endothelium-derived relaxing factor, a vasodila-
Shear sensitivity in animal cell culture Gooch and Frangos tor, while subsequently inhibiting the production of endothelin-1, a powerful vasoconstrictor [18]. The components of the signal transduction pathway, such as G proteins and phospholipases, are found not only in endothelial cells, but in all animal cells. Therefore, it is not surprising that other cell types such as osteoblasts and fibroblasts [19], and kidney tubule cells [20] are also stimulated by flow. Recent w o r k with Chinese hamster ovary cells cultured in suspension in a two liter bioreactor shows that these cells are also stimulated by mechanical forces [21"]. Although increased agitation intensity decreases cell viability, increased agitation induces the viable cells to proliferate more rapidly c o m p a r e d to low agitation control cultures. The increased proliferation rate was demonstrated b y flow cytometry, which revealed an increase in the rate of DNA synthesis and an increased fraction of viable cells in S phase. The proliferation rate remained elevated for at least six hours after the agitation rate was returned to control levels as demonstrated by a continued elevated rate of DNA synthesis and intrinsic cellular growth.
Conclusion Animal cells in culture are constantly exposed to mechanical forces. Excessive forces can cause cell death, though several practical and effective methods of protecting cells do exist. Smaller forces can produce more subtle, but important, changes in morphology, growth rate, and metabolic activity.
Acknowledgements This work was supported by the United States National Heart, Lung a n d Blood Institute Grant HL40696. JA Frangos is a recipient of the National Science Foundation Presidential Young Investigator Award. KJ G o o c h is a recipient of a National Institute of Health Biotechnology Training Fellowship.
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1.
PAPOLrrSAKIS E: F l u i d - M e c h a n i c a l D a m a g e o f A n i m a l Cells i n B i o r e a c t o r s . Trends Biotechnol 1991, 9:427-437. A review of theoretical models a n d experimental data of fluid mechanical d a m a g e as a result of particle-to-eddy, particle-to-particle, a n d cell-to-bubble interactions. For freely s u s p e n d e d cells grown in Ilfixed bioreactors, most cell damage is caused by bubble breakup or fast-draining liquid films around bubbles, while in microcarrier bioreactors d a m a g e is caused by interactions between microcarriers with other microcarriers and also with small turbulent eddies. 2.
LAKHOTIAS, pAPOUTSAKISE: Agitation Induced Cell I n j u r y i n M i c r o c a r r i e r c u l t u r e s . P r o t e c t i v e Effect o f Viscos-
ity is Agitation Intensity Dependent: Experiments and M o d e l i n g . Btotechnol Btoeng 1992, 39:95-107. 3.
BAVARIANF, FAN L, CHALME~ J: M i c r o s c o p i c VislmliTation of Insect Cell-Bubble Interactions. h RisIng Bubbles, A i r - M e d i u m Interface, and the Foam Layer. Biotechnol Prog 1991, 7:140-150.
4.
CHALMERSJ, BAVARIANF: M i c r o s c o p i c Visu~liTation o f Ins e c t Cell-Bubble I n t e r a c t i o n s . II: T h e B u b b l e F i l m and Bubble Rupture. Biotechnol Prog 1991, 7:151-158.
5.
CHERRYR, HULLE C: Cell Death in the Thin Films o f B u r s t i n g B u b b l e s . Biotechnol Prog 1992, 8:11-18.
6.
NEEDHAMD, TING-BEALL H, TRAN-SON-TAY R: A Physical C h a r a c t e r i z a t i o n o f GAP A3 H y b r i d o m a Cells: Morphology, Geometry, a n d M e c h a n i c a l P r o p e r t i e s . Biotechnol Bioeng 1991, 38:8384352.
7.
ZHANGZ, FERENCZI M, LUSH A, THOMAS C: A N o v e l Mic r o m a n i p u l a t i o n T e c h n i q u e f o r M e a s u r i n g t h e Bursti n g S t r e n g t h o f SIngle Mamm21i~*a Cells. Appl Microbiol Btotechnol 1991, 36:208-210.
8.
ZHANGZ, AL-RUBEAIM, THOMAS C: M e c h a n i c a l P r o p e r t i e s o f I i y b r i d o m a Cells i n B a t c h C u l t u r e . Bfotechnol Left 1992, 14:11-16.
RAMIREZO, MLrrHARASAN R: Effect o f S e r u m o n the P l a s m a M e m b r a n e Fluidity o f Hybridomas: an I n s i g h t into its Shear Protective Mecltanlsm. Biotechnol Prog 1992, 8:40-50. By measuring steady-state fluorescence anisotropy, it was determ i n e d that increasing s e r u m concentration, which is k n o w n to decrease shear sensitivity, decreased plasma m e m b r a n e fluidity. Cholesterol enrichment of the plasma m e m b r a n e also decreased plasma m e m b r a n e fluidity a n d shear sensitivity. 9.
10.
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GOOSEN M: Large-Scale I n s e c t Cell C u l t u r e . Curr Opin Biotechnol 1992, 3:99-104.
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HEBBEL R, MOHANDAS N: Reversible DeformationDependent Erythrocyte Cation Leak Extreme Sensitivity Conferred b y Minimal Peroxidation. Btophys J 1991, 60:712-715.
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Su W, CARAM H, HUMPHREY A: O p t i m a l D e s i g n o f the T u b u l a r M i c r o p o r o u s Membrane Aerator for ShearSensitive Cell C u l t u r e s . Biotechnol Prog 1992, 8:19-24.
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HSIEHH, LI N, FRANGOSJ: Shear-Induced P l a t e l e t - D e r i v e d Growth Factor Geue Expression In Human Endothelial Cells is M e d i a t e d b y P r o t e i n Kinase C. J Cell Physiol 1992, 150:552-558.
BERTHIAUMEF, FRANGOSJ: Flow-Induced P r o s t a c y c l i n Prod u c t i o n is Mediated by a P e r t u s s i s T o x i n - S e n s i t i v e G P r o t e i n . FEBS Lett 1992, 308:277-279. This paper presents data showing that fluid flow, like several hormonal agonists, induces prostacyclin production via a G protein d e p e n d e n t pathway. Flow potentiated the h o r m o n e - i n d u c e d production of prostacydin. 16.
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BHAGYALAKSHMIA, BERTHIAUME F, REICH M, FRANGOS J: F l u i d Shear Stress Stimmlates Membrane Phospholipid M e t a b o l i s m i n C u l t u r e d H u m a n E n d o t h e l i a l Cells. J Vasc Res 1992, 29:443-449.
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KUCHAN M, FRANGOS J; Shear Stress R e g u l a t e s E n d o t h e l i n - 1 R e l e a s e Via P r o t e i n K i n a s e C a n d cGMP
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REICH K, GAY C, FRANGOSJ: F l u i d S h e a r S t r e s s a s a Med i a t o r o f O s t e o b l a s t Cyclic A d e n o s i n e M o n o p h o s p h a t e P r o d u c t i o n . J Cell Phystol 1990, 143:100-104.
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KAWAHARAK, MATSUZAKIK: A c t i v a t i o n o f C a l c i u t n C h a n n e l b y S h e a r i n C t d t u r e d R e n a l Distal T u b u l e c e l l s . Biochem Biophys Res Commun 1992, 184:198-205.
21. •.
LAKHOTIA S, BAUER K, PAPOUTSAKIS E: D a m a g i n g &gitat i o n I n t e n s i t i e s I n c r e a s e DNA S y n t h e s i s Rate a n d A l t e r
c e l l - C y c l e P h a s e D i s t r i b u t i o n s o f CHO Cells. Bfotechnol Btoeng 1992, 40:978-990. A two-color flow cytometry m e t h o d was u s e d to determine the relative rate of DNA synthesis a n d t h e distribution of cells throughout the cell cycle. Increased agitation intensity decreases celt viability while simultaneously stimulating cells to proliferate more rapidly. This increased rate of proliferation lasted for at least six hours, even if agitation was returned to basal levels.
KJ Gooch a n d J A Frangos, 150 Fenske Laboratory, The Pennsylvania State University, University Park, PA 16802-4400, USA.