Biotechnol. Prog. 199 1, 7, 140- 150
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Microscopic Visualization of Insect Cell-Bubble Interactions. I: Rising Bubbles, Air-Medium Interface, and the Foam Layer Farshad Bavarian, L. S. Fan, and Jeffrey J. Chalmers* Department of Chemical Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43210
Through t h e use of microscopic, high-speed video technology, the interactions of two suspended insect cell lines, Trichoplusia ni (TN-368)and Spodoptera frugiperda (SF9), with air and oxygen bubbles were studied. Events such as cell-bubble attachment, cell-bubble collision, cell transport into the foam layer, and trapping of cells in the foam layer are presented and discussed. Based on these observations and those in a companion paper (Chalmers, J. J.; Bavarian, F. Biotechnol. Prog. 1991, following paper in this issue) and the experimental and theoretical work of other researchers, several mechanisms of cell damage as a result of sparging are presented.
Introduction The interest in large-scale growth of suspended animal and insect cells has increased greatly in the last 30 years. However, during this time it has been observed that cells can be damaged by hydrodynamic forces in the bioreactor. This hydrodynamic damage has been referred to as the “shear sensitivity”of the cells. It was initially believed that this damage was the result of the mechanical mixing in the system. However, more recent work by several researchers [e.g.,Tramper et al. (1986,1987,1988),HandaCorrigan et al. (1985, 19891, Oh et al. (19891, and Kunas and Papoutsakis (199011 indicates that the cell damage is most likely the result of cell-bubble interactions. There are three possible sites of cell-bubble interaction: at the sparger where the bubble is formed, as the bubble rises in the free medium, and at the medium-air interface. Tramper et al. (1987) have suggested that the shear stress associated at each of these sites would be sufficient to damage cells. They also suggested that if cells adhered to the medium-bubble interface, they would be subjected to high levels of shear stress as the bubble rises (Tramper et al., 1986). In an attempt to model the cell death rate, Tramper et al. (1988) assumed the existence of a hypothetical “killing volume” associated with each bubble and obtained reasonable agreement with experimental data. In 1987, on the basis of preliminary work, Tramper et al. suggested that the most probable region for cell damage was at the air injection region. Kunas and Papoutsakis (1990) and Handa-Corrigan et al. (1989)concluded that cell death takes place at the region of bubble disengagement at the medium-air interface. Kunas and Papoutsakis state, “Only when entrained bubbles interact with a freely moving gas-liquid interface, such as what exists between the culture medium and gas headspace, does significant cell damage occur”. Handa-Corrigan et al. (1989) based their conclusions partially on visual, microscopic observations of cells in a bubble column. On the basis of these observations, they further propose three possible mechanisms for this cell damage: (1) oscillatory disturbances caused by rapidly bursting bubbles, (2) physical shearing of the cells as film around a
* Corresponding author. 8756-7938/9 1/3007-0140$02.50/0
bubble drains, and (3) physical loss of cells in the foam. They also reported that they did not observe any direct cell-bubble interactions. However, with the equipment they reportedly used, both the field of view and depth of view would be very limited; consequently, we believe that an accurate observation of cell-bubble interactions would be limited. To overcome these limitations, we have developed a high-speed video (shutter speed up to 1/10000 s) and microscopic system with a relatively large depth and field of view to observe cell-bubble interactions. On the basis of these observations and the theoretical and experimental work of other researchers on bacterialbubble interactions and on bubble rupture, we propose several mechanisms for cell damage in a sparged bioreactor. In this paper we will report and discuss the observed interaction of cells with freely rising bubbles and with the air-medium interface and the presence of cells in the foam layer; in the second paper in this series (Chalmers and Bavarian, 1991),we will report and discuss the interactions of cells with bursting bubbles.
Materials and Methods Experiments were performed with the insect cell lines Trichoplusia ni (TN-368)and Spodoptera frugiperda (SF9). The cell history, method of cultivation, and medium used have been described previously (Goldblum et al., 1990) except in this work no protective medium additives, such as Pluronic or methylcellulose, were used. The cells in their late exponential phase of growth (i.e,, fourth day of growth) were used in all the experiments. In some experiments, the cell density was lowered by the addition of fresh TNM-FH medium. The cell density was measured with a hemocytometer and varied from 4.0 X lo5 to 1.3 X lo6 cells/mL. Figure l a shows the schematic diagram of the experimental apparatus. A Pulnix black and white video camera (Model TM-845, Pulnix America, Sunnyvale, CA) with adjustable electronic shutter speeds ranging from 1/60 to 1/10 000 s with 60 interline fields/s recording capacity was used to record images. The use of this camera with a asynchronous, high-speed shutter and high-reslution (800 horizontal pixels) VCR recorder was required for highspeed videography because of the high speed of rising
0 1991 American Chemical Society and American Institute of Chemical Engineers
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Biotechnol. Prog., 1991, Vol. 7, No. 2
a
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3/4" Video Recorder I
High Shutter Speed Video Camera
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B\IIlJ Microscope or Xlicro-Nikkor Len \ ~~
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Figure 1. Experimental apparatus for visualizing cell-bubble interactions. Panel a is an overview of the imaging system, while panel b is a schematic of the bubble column.
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Figure 3. Photographs of video images of a 1.7-mm air bubble rising through a suspension of TN-368 cells in TNM-FH medium. The large dark areas indicated by the arrow in panel b are clumps of cells adhered to the bubble.
A combination of objective lenses (i.e., 4X, 5X, and lox)
Figure 2. Photographs of video images of a 1.7-mm air bubble rising through a suspension of TN-368 cells in TNM-FH medium. The direction of bubble rise is from left to right in the photograph. In panel a the cells appear as small dark spots, while in panel b the cells appear as small white spots.
bubbles as a result of the magnification required to visualize cell-bubble interactions. The apparent bubble rise velocity is increased by the same factor as the magnification. The VCR recorder used was a Sony BVU 10 U-Matic videocassette recorder (Model VO-5800, Ichiomiya, Japan). The required magnification was obtained by using two different optical apparatus. For microscopic observations, the camera was mounted on an Olympus BMHJ metallurgical microscopewith dark-field and bright-field optics. The microscope objectives were positioned horizontally facing the surface of a specially designed bubble column.
and eyepieces (1.75X and 6.7X) enabled a wide range of magnifications,field of vision, and depth of field. However, for macroscopic observation of large air bubbles (i.e., 1-3 mm in diameter), a combination of a Nikon Micro-Nikkor lens (105 mm, f/2.8) and a Nikkon bellows extension connected to the video camera was used. By changing the length of the bellows, a range of magnification from 0.25X to 5X is obtained. Two methods were used to produce bubbles. For the macroscopic observations with the Micro-Nikkor lens system, air bubbles were injected through a 22 gauge hypodermic needle connected to a peristaltic pump. The air bubbles generated this way ranged in size from 1.5 X m in diameter. A low-power projector to 1.8 X light was used to illuminate the field of view. The size of the viewing field was varied from 13 X 9 to 4 X 3 (X10-3 m) depending on the bellows extension. At these low magnifications, individual features of the cells could not be seen. However, the macroscale interactions of cells were clearly observed. For microscopic observations, to better observe cellbubble interactions, oxygen microbubbles [ (50-150) X lo* m] were generated through the electrolysis of water. The bubbles were produced at the tip of the oxygen electrode (anode), which was placed in the bottom of the conical
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Figure 4. Photographs of video images of cells (indicated by arrows) attached to oxygen bubbles. The bubbles appear as black spheres, while the cells appear as a lighter shade. Panels a and b are of SF-9 cells in TNM-FH medium, while panels c and d are of TN-368 cells in TNM-FH medium. The camera shutter speed was 1/10 000 s, and the line divisions correspond to 202 X lo*, 202 X lo*, 180 X lo*, and 102 X lo* m in panels a, b, c, and d, respectively.
section of the reactor (see Figure lb), thus preventing the sedimentation of the cells in the dead zone of the reactor. The voltage and the amperage were held constant a t 15 V, and 4.5 mA, respectively. The hydrogen electrode (cathode) was placed near the anode; however,the chamber for the cathode was isolated by way of equipment design such that the microscopically observed cells were not in contact with the H2 bubbles. This ensured that only 02 bubbles were observed interacting with cells. The rectangular bubble column used in these microscopic imaging experiments was made of a 3.2 X thick acrylic surface with 0.5 X m thick Lexan spacing sheets. The final thickness of the column with the adhesives was maintained at 1 X m. The overall dimensions of the column was 50 X 10 X 0.1 m) with sampling ports at 1.0 X lo-* m intervals. The small column thickness provided a higher ratio of the depth of focus to the gap thickness producing sharper images. Low-power cold projector light was used to focus light on a small field of m). The column was placed on a moving view, 7 X 4 ( table so that different positions inside the column could be observed by moving the table in lateral or axial directions. The column used for macroscopic observations of the larger, air bubbles was similar to the previous column with the exception of an increase in the thickness, increased to 3.2 X m, and the removal of the chamber for the hydrogen electrode.
Results The observed cell-bubble interactions reported in this paper can be classified into three categories: cell(s) interacting with freely rising bubbles, cells at the mediumair interface, and cells trapped in the foam layer. The visual observations presented in this paper are considered representative of over 30 h of analysis of video images made of experiments conducted with this system. When figures are presented of a cell(s) attached to a bubble, the
analysis of several video frames before and after the one shown conclusively proved that the cell(s) were attached to the bubble. Cell-Bubble Attachment to Freely Rising Bubbles. Air Bubbles. Initially, 1.7-mm air bubbles were injected into the 3.2 X m thick, two-dimensional reactor. As mentioned previously, the rise velocity of bubbles, which is increased by the same factor as the magnification, necessitated the use of the low-magnification Nikon bellows system (5X). However, the use of lower magnifications limits the observations of individual cell-bubble interactions and only macroscopic behavior of the suspended cells can be observed. Figure 2 shows photographs of a representative video image of an air bubble rising through growth medium containing TN-368 cells. The roughness that exists around the bubble surface, especially a t the edge of the bubble, where the camera is focused, is believed to be cells adhered to the bubble. Figure 3 shows the attachment between large cell clusters (>lo0 cells) and an air bubble. The cell clusters are the dark areas under the rising bubble indicated by an arrow in panel b. In these experiments, the bubble injection was stopped for a short period to let the cells settle in the conical section of the injector. The air bubbles were then injected through the settled layer of cells, ensuring the contact between the cells and the bubble surface. The images were taken after the bubble had risen 15 X m from the point of injection. From these images there is no doubt that the large cell cluster attached to the bottom of the bubble is adhered cells. OxygenMicrobubbles. To better visualize individual cell-bubble interactions, smaller oxygen bubbles were used. These bubbles ranged in size from 2 to 12 (XlOW5) m in diameter. In experiments using this method of bubble production, the generation of bubbles was constant and at a sufficient rate to maintain the cells in suspension. Figure 4 illustrates a series of individual cell-bubble
Biotechnol. Prog., 1991, Vol. 7,
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Figure 5. Photographs of a series of video images of a bubble colliding with a small group of TN-368 cells. The line divisions in the image correspond to 180 X lo4 m and the shutter speed was 1/10 000 s.
attachments of TN-368 (panels a and b) and SF-9 (panels c and d) cells in TNM-FH medium. With these smaller bubbles it is possible to use higher magnification and, consequently, clearly observe individual cell-bubble attachment. Approximately 5-10 % of the rising bubbles had cells attached to them. In none of our observations did we observe any cell becoming detached from a bubble, indicating that the strength of the cell-bubble attachment is sufficient to withstand the drag force acting on the cell. Most of the attachments of TN-368 cells to the bubbles appear to be through the cells' appendages. After extended periods of sparging, TN-368 appendages become thinner, possibly signifying the elongation of the cells' appendages and alteration of the cells' morphology. Individual Cell-Bubble Collision. Figure 5 shows a time series of a cell-bubble collision. The cells shown here are within a streamline flow profile around the rising
143
bubble. The small bubble size and their measured rising velocities suggest that the bubbles are well within the Stokes' regime (see Discussion). Therefore, the cells approximately follow the streamline flow around the bubbles. In some cases we have observed cells tumbling and rotating around bubbles, which is probably the result of portions of the cell positioned on different streamlines. Figure 6 contains photographs of a video sequence showing the attachment of a SF-9 cell to a bubble. While an image of the initial cell-bubble contact is not available, the movement of the cell from Oo (relative to the direction of flow) to 180' is clearly shown. Once the cell reaches 180°, the cell stops moving relative to the bubble and remains attached as the bubble rises. While the image of the initial collision is not available, on the basis of the movement of the cell on the bubble surface it is reasonable to assume that the collision occurred just before the first image in the sequence. Attachment of Cell Cluster to Multiple Bubbles. Figure 7 shows examples of the attachments between cell clusters and microbubbles. The large size of this cluster, containing more than 200 cells, and its configuration can promote a different mode of attachment to bubbles. For instance, as shown in Figure 7, small bubbles can become adhered/trapped in the cell cluster. The occurrence of these large clusters of cells is not unusual; Hink and Strauss (1979) reported that the addition of methylcellulose to the growth medium can prevent this clumping phenomenon in suspension culture in spinner flasks and bioreactors. Cells at the Air-Liquid Interface, Cells within the Foam Layer, and Transport into the Foam Layer.The abundance of cells attached to the air-medium interface at the top of the column is indicated in Figure 8, panels a and b, while panels c and d shows cells trapped in the foam layer. Two mechanisms of cell transport into the foam layer are demonstrated in Figures 9 and 10. In Figure 9 a clump of cells attached to the interface is observed to become covered from beneath as bubbles cover the clump, while in Figure 10 cells are observed to be pulled into, and disappear within, the foam layer as a group of bubbles enters the foam layer.
Discussion The images in the preceding section present a summary of our observations of cell attachment to freely rising bubbles and to the air-liquid interface. Based on these observations, several possible mechanisms for cell death can be proposed. In this section we will discuss the probable mechanisms of cell-bubble attachment and possible mechanism of cell death from the observations reported above. Cell-Bubble Adhesion. To the best of our knowledge, there exist no reported cases of insect or mammalian cells adsorbing to bubbles (risingor stationary). However,there exist many reported cases of bacteria adsorbing to rising bubbles and aerosols produced from bursting bubbles (Blanchard and Syzdek, 1978, 1982; Bezedek and Carlucci, 1974; Weber et al., 1983). The mechanisms of bacterial adhesion to gas-liquid interfaces have been reviewed by Kjelleberg (1984, 1985). The mechanisms appear to be primarily the result of cell surface hydrophobicity (Hermansson and Dahlback, 1983); however, other mechanisms such as interactions of cells with lipids adsorbed on the interface (Kjellebergand Stenstrom, 1980) may also be important. Parker and Barsom (1970) reported that the surface microlayer of seawater, the very thin layer of liquid a t a liquid-air interface, contains 4 times as much algae and
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Figure 6. Photograpns or a series or video images ot a SP’-8cell colliding and becoming attached to a bubble. ‘l’hearrows in panels a and b indicate the initial position of the cell. The line divisions in the image correspond to 202 X lo4 m.
protozoa as the liquid taken 1.0 X 10-l m beneath. They also report that the surface microlayer of freshwater habitats can contain 5 times as much dry weight of total particles as compared to a sample taken 10 X m beneath (the size of the particles was greater than 0.2 X m, and they were most probably microorganisms). Probably the most commonly used method of defining these hydrophobic interactions is associating a change in free energy, AG, with the process of bringing a cell and an interface together from an infinite separation. This change in AG can be related to the interfacial tensions of the surfaces involved. Such a model was developed by Absolom e t al. (1983) for bacterial adhesion. As predicted by their model, and confirmed by experimental work, changes in the interfacial tension of the suspending liquid, and thereby changes in AG, can significantly change the amount of bacteria that adheres to surfaces.
Hydrophobic interactions have also been reported in the adhesion of eucaryotic cells to surfaces. Malmqvist et al. (1984) reported that mouse myeloma (SP/O) and Chinese hamster ovary (CHO) cells have significant hydrophobicity and that the degree of hydrophobicity can also be altered as a result of different medium components that change the surface tension of the medium. We have observed (Chalmers et al., 1988) that low levels of laminar shear stress result in aggregation of TN-368 cells. These aggregations are very similar to the cell aggregates reported in this work and are believed to be the result of hydrophobic interactions. It is interesting to note that the addition of Pluronic F-68, which lowers the surface tension of the medium, decreases this aggregation. While it seems likely that the cell-bubble attachment is the result of hydrophobic interactions, it is possible that another mechanism such as hydrostatic charge is also
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Figure 7. Photographs of a series of video images, panels a-d, of two 02 bubbles (arrow A) colliding and becoming attached to a large clump of TN-368 cells (>200 cells). Note that the 02 bubble indicated by arrow B is attached to the clump by a thin TN-368 appendage. Panels e and f show another example of 02 bubbles attached to a clump of cells.
involved. The fact that the cells adsorb to both air bubbles and oxygen bubbles seems to rule out the possibility of there being a unique effect as a result of oxygen bubbles generated through electrolysis. Figure 6 represents a probable mechanism by which a cell-bubble adhesion takes place as a result of a cell-bubble collision. This mechanism is similar to the bubble-particle interactions theory developed for ore flotations. Kelly and Spottiswood (1982), on the basis of an extensive review of the literature, reported that "the rate of (ore) flotation is equal to the product of three factors: (1)the rate of collision between particles and bubbles, (2) the probability of adhesion, and (3) the probability that the adhering particle will not be subsequently detached because of turbulence". These three probabilities have been combined in asingle
equation by Sutherland (1948):
[
p = [~TR,R,vC] sech - p ,
(1) 4R1 3 ~ ~ 1 The first term in brackets corresponds to the probability of collision, the second term corresponds to the probability of adhesion, and p s corresponds to the probability of formation of a stable bubble-particle attachment. The variable p, the induction time, is the minimum time interval required for cell-bubble contact to ensure that adhesion is successful. Therefore, not every cell-bubble collision may result in a cell-bubble attachment. This theoretical relationship has been shown to correctly predict the experimental result of ore flotation. It is interesting to note that the adhesion of ore to bubbles is governed by 2
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Figure 8. Photograpns or video images, panels a and b, of cells attached to the air-medium interlace a t the top of the column. Panels c and d are photographs of cells trapped in the foam layer above the medium. The line divisions correspond to 180 X lo*, 180 X lo4, 180 X lo*, and 102 X lo* m in panels a, b, c, and d, respectively.
interfacial forces, of which hydrophobic interactions are the most significant. Therefore, it is possible that the current theory developed for kinetics of the flotation process can be modified with bubble-medium-cell surface parameters to account for accumulation and entrainment of cells to the top foam layer. Shear Damage Mechanism of Cells Attached to Bubbles. Table I presents representative values of experimentally determined bubble rise velocities as compared to the theoretical rise velocities based on Stokes’ law. In all cases, the Reynolds number was less than 1. Since TNM-FH medium contains a large amount of proteins, which act as surfactants, it can be assumed that the bubbles in the bubble column can be approximated as rigid spheres, as is required in Stokes’ law. As expected, the actual bubble rise velocity varied around the theoretical value. Since the measurements were made from a fixed frame of reference, bulk movement of fluid could be present as a result of other bubbles rising through the medium, thereby increasing or decreasing the apparent bubble rise velocity. Note that the presence of a cell adsorbed to the bubble apparently does not significantly affect the bubble rise velocity. Assuming a flow regime in which Stokes’ law applies, the shear stress on a surface of a sphere can be determined from (Denn, 1980)
Based upon this relationship, the theoretical and experimental values of the bubble rise velocities, and the viscosity of TNM-FH medium, the maximum shear stress (90’ to the direction of flow) on the surface of the bubbles listed in Table I is presented in Table 11. In a previous article (Goldblum et al., 1990), it was reported that a level of shear stress of 0.12 N/m2 in a cone and plate viscometer will result in a rate of lysis of 0.113 %
LDH released/s for TN-368 cells in TNM-FH medium without protective additives. In other words, it can be expected that 34% of the cells would be lysed after being subjected to 0.12 N/m2 for 300 s. The length of time for the bubbles in Table I to rise to the top of the column would range from 17 to 270 s. While the level of shear stress that a cell adsorbed to a bubble experiences is not necessarily the same as the theoretical surface shear stress described by eq 2, it is reasonable to assume that cells adsorbed onto a rising bubble would be subjected to potentially lethal levels of shear stress. As further evidence for the detrimental effect of shear stress as a result of being adsorbed to a rising bubble, many of the adsorbed cells that we observed had a rounded morphology. This rounded morphology, unlike the normal morphology, which included several appendages, is also observed on nonlysed cells after being subjected to comparable levels of shear stress in a cone and plate viscometer (Chalmers et al., 1988). Several assumptions were made in the above discussion that need to be addressed. First, the level of shear stress on the surface of a sphere that is rising with the indicated velocity is not necessarily the shear stress that a cell adsorbed onto the bubble would experience. Two extremes will be considered to demonstrate the accuracy of this m bubble listed in Table assumption. For the 6.6 X I, the cell adsorbed to the bubble was relatively small, 0.9% of the bubble volume, and was located a t approximately 158’ (upper drawing in Figure 11) to the direction of flow on the bubble. While the cell would slightly deform the fluid streamlines around the bubble, it is reasonable to assume that the cell would experience a surface shear stress approximately the same as the bubble a t that location on the bubble surface. This corresponds to a value of 0.045 N/m2. m bubble listed in On the other hand, the 8.1 X Table I had a 15 X lo4 m cell adhered to it. In this case
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without being attached to a bubble. Since the drag force consists of the form drag and the frictional drag, we can assume that surface shear stress on the cell’s membrane would be 50-60% of the value of the surface shear stress for an individual sphere of the same size. The maximum shear stress on a 15 X lo4 m sphere, at an infinite distance m/s is 0.85 from another sphere, moving at 3.54 X N/m2. Therefore, the actual value of the maximum surface shear stress that the cell membrane would experience is 0.425 N/m2. A second assumption was that the shear stress that a cell would experience in a cone and plate viscometer is comparable to the shear stress on the surface of a bubble (or cell) as it rises through the medium. As discussed by Martin et al. (1979), the bulk shear stress that is created in a couette and/or cone and plate viscometer is not the same shear stress that the cell experiences. Unfortunately, the accurate, quantitative determination of the actual stress on the cell membrane is complex and beyond the scope of this work; however, with the assumption that the cell can be modeled as a rigid spheroid, the following relationship can be used to estimate the actual shear stress applied to the cell membrane: =
sin ( ~+ te,)]
T , ~ 5 / 2 [ ~ ~
Figure 9. Photographs of a series of video images of a clump of cells attached to the bottom of the foam layer as it is being trapped by rising bubbles from underneath.
the cell was attached to the bubble by an appendage and was located 180’ from the direction of motion (lower drawing of Figure 11). Stimson and Jeffrey (1926) have solved the Stokes’ flow problem around two spheres of radii r1 and r2, separated by a center distance of d. If we assume that the bubble and cell are separated spheres, we can use their solution to determine the effect that the presence of the bubble has on the force needed to maintain the motion of the cell, i.e., the drag force. They represented this modified drag force as
F = 6xpRUX (3) where A is the correction factor for the effect of one sphere on the other one. For a cell of 15 X lo4 m diameter and a center distance slightly greater than the sum of the bubble and cell radius, X has a value of approximately 0.5-0.6. Therefore, the drag force on the cell is 50-60% of the drag force that would exist if the cell were traveling at the same velocity
(4)
As can be observed from this equation, the shear stress on the cell membrane oscillates sinusoidally with a maximum equal to 2.5 times the bulk shear stress. Therefore, a cell being subjected to a bulk shear stress of 0.12 N/m2 in a cone and plate viscometer would actually be subjected to an oscillating shear stress with a maximum of 0.3 N/m2 and a period of 3.14 X s. While it is difficult to compare the effect of this oscillating shear stress in the cone and plate system to the constant shear stress that a cell would experience attached to a rising bubble, the magnitude of the shear stress is comparable. It should also be noted that these calculations were based on oxygen microbubbles in a Stokes’ flow regime. In a conventional bioreactor the bubbles are generally much larger. Bubbles with a Reynolds number of about 120 ( ~ 1 - 3mm in diameter) exhibit complex hydrodynamic behavior since the flow around the bubble falls between essentially two hydrodynamic regimes. The high surfactant condition existing in the medium further complicates the flow around the bubble. As reported by Andrews et al. (1988), at high surfactant concentrations a third hydrodynamic regime for these bubbles was observed, which was referred to as “large-wake” hydrodynamics. In this regime the high concentrations of surfactant alter the boundary condition around the rising bubbles such that the flow separation is moved to the front stagnation point, thus creating a large wake around the rising bubble. The photographic evidence of this phenomena is reported by Subramanian and Tien (1970). The implications of the large-wake hydrodynamics are very significant for cell capture efficiency by bubbles. As opposed to eq 1,which is based on the potential flow around the bubble, in large-wake hydrodynamics cell-bubble collision or cell-bubble attachment is not required for a cell to be carried to the top air-liquid interface. For instance, a recirculating wake closer to the equator of the rising bubble can trap cells whose trajectories would take them past the bubble (Andrews et al., 1988). The largewake hydrodynamics basically implies a higher rate of cell entrainment to the foam layer, resulting in a larger physical loss of cells. Loss of Cells in the Foam Layer. As shown in the previous section, cells are frequently trapped in the foam
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Figure 10. Photographs of a series of video images of a line of cells attached to a rising bubble as they are pulled into the foam layer. Table I. Calculated and Theoretical Bubble Rise Velocities of Bubbles Rising in the Bubble Column
bubble diameter, ~ 1 0m 6
experimental rise velocity, XlO4 m/s
theoretical rise velocity, x104 m/s
29O 36 46O 66b 74 81b 110
5.6 0.81 9.5 22 16 35.4 67
3.7 5.9 9.5 20 25 29 56
a A 23-pm TN-368 cell was adsorbed to the bubble. A 13-19-pm SF-9 cell was adsorbed to the bubble.
layer, contributing to a net loss of viability by two possible mechanisms. First, the trapped cells in the foam are considered physically lost since they are not usually recovered from the foam. Second, cells are subjected to
Table 11. Maximum Shear Stress (90' to the Direction of Flow) on the Surface of a Sphere Assuming Stokes Flow
sphere diameter, um 29 36 46 66 74 81 110
calculated surface shear stress. N/m2 0.070 0.081 0.074 0.12 0.078 0.15 0.219
theoretical surface shear stress, N/m2 0.046 0.056 0.074 0.11 0.12 0.134 0.18
inhibitory environmental conditions such as possible dehydration and nutrient depletion in the foam. Once cells are attached to the air-liquid interface, cells can become trapped in the foam layer by several mechanisms: (1)net movement of an individual cell attached to a bubble as the bubble enters the foam layer, (2) net movement of a cell cluster attached to a bubble(s) as it
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149 Oxygen Bit hhle
t
Direction of Buhhle Rise
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Oxvgen Bubble
Figure 11. Scale drawing representing the relative size and
position of cells observed adhered to rising bubbles.
(they) enter the foam layer, and (3) the bulk movement of cell(s) either attached to a bubble or to the interface as the cells are covered from underneath by succeeding bubbles. Conclusion This preliminary work suggests two possible mechanisms for cell damage in a sparged bioreactor: (1) the adsorption of cells to rising bubbles and the associated shear stress on the cell membrane as the bubble rises and (2) the attachment of cells to the air-medium interface and the transport of these cells into the foam layer. In the next paper of the series (Chalmers and Bavarian, 1991), two other possible mechanisms based on observations of the interactions of cells with bursting bubbles will be presented. While the first mechanism is probably not the primary method of cell damage, especially when compared to (2) and the mechanisms proposed in the next paper, it still might contribute to cell death observed in bioreactors. The adsorption of cells to bubbles is probably the result of hydrophobic interaction between the cell and bubble. Researchers have reported that by changing the properties of the growth medium, such as raising or lowering the surface tension, the amount of bacterial adhesion to surfaces can be changed. It is possible that the reported protective effect of serum and surfactants, which have been shown to lower the surface tension of the growth medium, are the result of decreasing the number of cells that adhere to bubbles and thereby preventing cell damage. Current work in our laboratory is investigating this hypothesis. Notat ion 0 induction time x correction factor CL viscosity probability of particle attaching to rising bubble P probability of formation of stable bubble-particle Ps attachment Y bulk shear rate 0 angle relative to bubble rise direction R1 radius of bubble radius of particle or bubble R2 Tro tangential shear stress V relative bubble-particle velocity AG change in free energy
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Tramper, J.; Joustra, D.; Valk, J. M. Bioreactor design for growth of shear-sensitive insect cells. In Plant and Animal Cell Cultures: ProcessPossibilities; Webb, C., Matvituna, F., Eds.; Ellis Horwood Limited: Chichester, England, 1987; pp 125136. Tramper, J.; Smit, D.; Straatman, J.; Valk, J. M. Bubble-column design for growth of fragile insect cells. Bioprocess Eng. 1988, 3, 37-41. Weber, M. E.; Blanchard, D. C.; Syzdek, L. D. The mechanism of scavenging of waterborne bacteria by a rising bubble. Limnol. Oceanogr. 1983, 28, 101-105. Accepted January 15, 1991.
