JP2001521736A - How to enhance mixing in a rotating bottle - Google Patents

Abstract

  (57) [Summary] A method is disclosed for enhancing the mixing of materials in a rotating jar by introducing controlled turbulence in axial and cross flow. The effectiveness of the method is demonstrated by the introduction of controlled flow perturbations during processing to achieve increased efficiency of cell culture and virus growth in rotating jars.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION In the fields of pharmacy, biochemistry and medicine, manual and fully automated rotations for processes such as cell growth, infection, production of heterologous glycoproteins, preparation of vaccines and high-density culture of plant cells. Bottle systems have been used for over 40 years [H. Tanaka, F .; Nis
hijima, M .; Suwa, and T.W. Iwamoto, Biotechn
ol. Bioeng. 25, 2359 (1983); Tanaka, Pro
cess Biochem. Aug. , 106 (1987); Y. Hong
, T .; P. Labuzza, and S.M. K. Harlander, Biotec
hnol. Prog. 5: 4,137 (1989); A. Elliot, B
ioprocess Tech. 10, 207 (1990); G. FIG. Kalt
hod, Novel Carrier and Reactor for Culture of Attachment Dependent Mammalian Cells. D. SC. Thesis. Washington Uni
version, St .; Louis, Missouri (1991); I.
Tsao, M .; A. Bohn, D .; R. Omstead, and M.S. J. Mu
nster. Annals N.A. Y. Acad. Sci. 665, 127 (19
92); Pennell and C.I. Milstein, J .; of. Im
mun. Meth. 146, 43 (1992); Olivas, B .; B. D
. -M. Chen, and W.C. S. Walker, J.M. Immun. Meth
. 182, 73 (1995); Singhvi, J .; F. Markusen
, B. Ky, B .; J. Horvath, and J.M. G. FIG. Aunins, Cyt
technology 22, 79 (1996); Kunitake, A
. Suzuki, H .; Ichihashi, S .; Matsuda, O .; Hira
i, and K. Morimoto, J .; Biotechnology 52:
3,289 (1997). ]. Despite the efforts of many researchers to develop systems based on unit operations, such as microparticle carrier culture, to produce anchorage-dependent cells or cell products [E. Van Hemert, D .; G. FIG. Kilburn
, And, A .; L. Van Wezel, Biotechnol. Bioeng
. 11, 875 (1969); Horn and W.M. McLimans
, Biotechnol. Bioeng. 17, 713 (1975); E. FIG.
Spier and J.M. P. Whiteside, Biotechnol. B
ioeng. 18, 649 (1976); W. Levine, S.M. Wang
, And W.S. G. FIG. Tilly, Biotechnol. Bioeng. 2,
821 (1979); J. Clark and M.S. D. Hirtenst
ein, J. et al. Interferon Research 1,391 (1981)
B.); J. Montagnon, B .; Fanget, and A. J. Nic
olas, Developments in Biological Stan
dards 47, 55 (1981); G. FIG. Edy, Adv. Exp. Me
d. Biol. 172, 169 (1984); Rivera, C.I. G. FIG. Sj
Osten, R.A. Bergman, K .; A. Karlsson, Research
h in Veterinary Science 41, 391 (1986)
R .; M. Gallegos Gallegos, E.L. L. Espinosa
Larios, L .; R. Ramirez, R .; K. Schmid, and A.S.
G. FIG. Setien, Archives in Medical Research
h 26: 1, 59 (1995). The rotating bottle system is still mainly used between researchers and traders. In addition, when producing cell culture products (ie, vaccines) on an industrial scale, even if a unit-based system is used, the cell bottle must be rotated before moving to the microparticle carrier culture in the final growth stage. Culture is frequently used [V. G. FIG. Edy, Adv. Exp. Med. Biol. 172,
169 (1984)].

[0002] There are several possible reasons why the use of rotating bottles is so popular. The most notable reason is that processing relies on very simple techniques. Approximately 1 /
Fill to 3 and rotate in the axial direction. Therefore, scale-up development is not required, industrial development time is shortened, and new products can be introduced to the market more quickly. The system keeps the fluid-gas in contact at all times and can easily add nutrients without interrupting the process. In addition, accurate control of nutrient and waste levels as the process can be maintained under sterile conditions for extended periods of time and contamination of one or more jars does not contaminate the entire lot. And direct monitoring of cells is relatively easy [E. I. Tsao, M .; A. Bohn, D .; R. Omste
ad, and M.D. J. Munster. Annals N.A. Y. Acad. S
ci. 665, 127 (1992)].

[0003] On the other hand, in a rotating bottle, the surface area available for propagation is small and the amount of harvested fluid obtained is small. Since hundreds of spinning bottles are typically operated in a single production run, requirements such as manpower and equipment location are more stringent than unit operating systems such as particulate carriers. A new fully automatic system addresses this problem [R. Kunita
ke, A .; Suzuki, H .; Ichihashi, S .; Matsuda, O .;
Hirai, and K. Morimoto, J .; Biotechnology
52: 3,289 (1997); Archer and L.A. Wood,
Proceedings of the 11th Annual Meeti
ng of European Society for Animal Ce
ll Technology, Brighton, U.S.A. K. , Septemb
er 2-6, 1991. ]. In addition, cell growth and infection performance is likely to be significantly reduced, probably because adherence of infected cells to host cells attached to the bottle wall is arrested, probably by flow and mixing dynamics [Y. A. Elliot,
Biopress Tech. 10, 207 (1990)]. While these issues clearly demonstrate the need for flow analysis and process design criteria, no suggestions for a solution to any of these problems have been published to date.

[0004] Conventional rotary bottle mixing methods are not efficient. In the case of cell seeding, cell growth and / or virus growth, the bottle is generally rotated in one direction at a uniform speed. Typical speeds are between 0.125 rpm and 5.0 rpm. However, with this uniform rotation, cells or other particles, such as viruses, are trapped in circular orbits, creating a dead zone inside the jar that can never rise to the surface of the jar. For cell seeding,
For anchorage-dependent cells, it is important that the cells quickly contact the side wall of the spinning bottle. This is because such cells can form a cell layer only when they adhere to the container wall. Slow attachment results in reduced cell viability and / or inhomogeneous seeding, resulting in uneven growth of the rotating jar surface. Such inefficient mixing during cell growth can be caused by poorly mixed media supplying appropriate nutrients (eg, oxygen) to the cells as the bottle rotates and toxins (
Since, for example, carbon dioxide cannot be removed, cell proliferation is suppressed. For many viral growths, it is important that the virus attach quickly to the cells in order to maintain the infectivity of the inoculum and achieve a rapid and homogeneous infection. Also, these goals cannot be achieved when the mixing of the liquids is not sufficient.

[0005] The disadvantages of conventional rotary bottle mixing will be understood when considering their use in virus propagation, especially when the virus inoculum is used in the form of an infected cell suspension. Such cases include several herpesviruses such as the chicken marrakech virus (D. Ben Nathana and S. Lustig "Pr
"action of Marek's Disease Vaccine"
, Viral Vaccines, Wiley-Liss, 1990, pp. 146-64. Three
47-365) and Varicella Zoster Virus (P.
J. Provost et al., U.S. Patent Nos. 5,360,736 and 5,607,8.
No. 52; Krah et al., "Enhancement of Varicella-
Zoster Virus Plaquing Efficiency wit
Han Agoverse Overlay Medium "J. Vir. Me
foods 27, pp. 319-326 (1990). ). Virus growth efficiency depends on the infectious cells in contact with the cell layer forming the foci. The infection spreads from these nests to the cell layer. However, in conventional spinning bottle mixing, many infectious cells are captured in closed symmetric orbitals and never reach the cell layer on the bottle surface. Furthermore, the conventional rotary bottles have a large inhomogeneous region due to insufficient axial mixing.

In this application, the mathematical and experimental characterization of fluid flow profiles inside a rotating spinning bottle, such as particle trajectories, fluid mixing patterns, and unsteady state flow plans, have been discussed in detail. . The results suggested a method whereby optimal growth and infection of cells could be obtained by simple modification of the rotation of the spinning bottle. Thus, the present invention describes an improved method of mixing in a rotating bottle to introduce cross and axial flow disturbances. These perturbations disrupt the closed trajectory where cells are captured during conventional mixing and promote particle settling. The present invention further relates to a mixing method which ensures that the cells are in contact with an appropriate amount of nutrient-rich medium and enhances the contact of the cells with the inner wall or cell layer of the rotating bottle, thereby increasing the productivity.

SUMMARY OF THE INVENTION [0007] The present invention is directed to a method for enhancing mixing in a rotary bottle by introducing controlled disturbances in axial and cross-directional flow. The effectiveness of the method is demonstrated by achieving improved cell culture and virus growth efficiency in a spinning bottle.

DETAILED DESCRIPTION The present invention is directed to a method for enhancing the mixing of materials in a rotating bottle comprising controlled disturbance of axial and cross flow.

In an embodiment of the method according to the invention, the controlled disturbance of the flow comprises the following groups: (a) uniform rotation of the rotatable bottle with oscillating movement, (b) time-dependent rotary speed of the rotatable bottle, (C) the time-dependent rotation speed of the rotating bottle with oscillating motion, (d) the time-dependent rotating direction of the rotating bottle, (e) the time-dependent rotating direction of the rotating bottle with oscillating motion, (f) the time A time-dependent rotation speed of the rotating bottle, which combines the dependent rotation direction and the swinging motion.

The term “material” refers to a cell culture and the media required to support the culture,
Also, solutions or suspensions of liquid and solid materials, such as virus-infected cells and media required to support these cells.

[0011]Uniform rotation of rotary bottle with oscillating motion  One mode of enhancing mixing and settling is to combine rotation and rocking.
use. The axis of rotation of the jar is the line connecting the center of the jar along the length of the jar.
Is defined as The oscillating movement is a sweep of the rotating bottle about an axis perpendicular to the rotating axis of the rotating bottle.
It is defined as the pull angle. This angle can range from about 0 degrees to about +20 degrees or -20 degrees.
The rotation speed is about 0 to about 50 rpm, and the frequency of swing versus rotation is about 0
-About 31.4. Preferred conditions for the combination of rocking motion and bottle rotation are about +1
Oscillation angle from 0 degrees to about -10 degrees, defined by frequency of oscillation versus rotation of about 1.8
You.

[0012] The wobble makes the flow geometry time dependent and thus necessarily disrupts the cell motility pattern. It is believed that this method has the greatest effect. The computation method for this problem would be quite complicated. Fortunately, the method can be easily studied by experiment.
Shaking greatly enhances mixing in the jar. This is shown in FIGS. 5 and 6, which tested the mixing of glycerin with a pH indicator (Bromthymol Blue). First, add glycerin to the indicator and 0.5 m
L of 1M NaOH. As a result, glycerin turned deep blue. Place the spinning bottle on the device and remove about 20 mL of glycerin from the bottle with a syringe, 1.0 mL
Upon mixing with mL of 1 M HCl, the fluid turned yellow. This fluid was then refilled near the rear wall of the jar and mixed with the fluid at the bottom of the jar with a specially modified spatula.
Next, the rotating bottle was rotated. In experiments with real cells, the choice of rocking frequency will be limited by consideration of immersing the cells in the nutrient medium as evenly as possible.

FIGS. 21 (a)-(c) show a control experiment without rocking. FIG. 21 (d)-
(I) shows two different rocking frequencies, 1.6 and 3.2 revolutions per rocking cycle. Without rocking, half of the bottle is yellow after 32 rotations and the blue-yellow interface hardly moves after 64 rotations. In contrast, when rocking is used, the fluid after 64 revolutions at the above two frequencies has completely turned yellow, indicating that complete mixing of the system has been achieved. These results indicate that the swing
Demonstrates that it actually enhances mixing by disrupting ring cell motility and enhancing cell sedimentation.

The oscillating motion can be introduced in a number of ways including, but not limited to, introducing the oscillating motion to a roller holder (FIG. 22) or providing a protrusion on a roller or rotating bottle (FIG. 23). . The rack of the roller holding machine as shown in FIG. 22 can change the frequency of the swing versus the rotation, the swing angle, the number of rotations and the rotation speed of the bottle extremely freely. The present invention further contemplates the use of a single level roller retainer.

[0015]Time-dependent rotation speed of rotating bottle  Another method of disrupting the periodic cell trajectory is to operate the bottle in stop-go mode
That is. The bottle movement consists of two alternating parts. Bottle is time t₁During rotation at constant speed U, time t_TwoInside is stationary. In the second part of the flow cycle, the cells sediment at the final velocity. The purpose of using this method is to disrupt the periodic cell trajectory,
It is to plan. This preliminary result also suggests that this mode of exercise will increase subsidence,
The degree was shown to strongly depend on the sedimentation velocity.

Another method is to vary the rotation speed as a function of time. This method was applied to a varicella vaccine production method. This method has been used up to 0.2
A rotation speed of 5 rpm was used. Experiments were performed using a higher rotation speed during the cell expansion phase and a lower rotation speed during the first 6 hours of the infection phase. As described below, the slow rotation speed during the viral growth phase allowed the varicella-infected cells to reach the inner surface of the spinning bottle more quickly, resulting in improved virus production levels. In addition, the use of rotations faster than 0.25 rpm during the cell expansion phase improved cell growth efficiency.

In previous experiments, the growth efficiency of anchorage-dependent cells and cell culture products (ie, varicella virus) in rotating bottles was inferior to the growth efficiency observed in stationary cultures such as T-type culture bottles. Proved to be. One reason for these differences was speculated to be that the circulating liquid stream capturing the particles in a circular orbit slows down the sedimentation rate of cells (uninfected and infected cells) present in the spinner culture. . As a result of the reduced sedimentation time, the efficiency of cell plating of uninfected cells was reduced, and the degradation time of virus-uninfected cells that could not reach and / or infect the cell culture surface was extended.

[0018] The sedimentation and attachment kinetics of virus-infected cells were studied at various rotation speeds. In addition, the attachment and growth of cells to the surface of the spinning bottle was examined to elucidate the primary mechanism controlling the observed differences in cell growth rate as a function of rotation speed.

[0019]Time-dependent rotation speed of a rotating bottle with oscillating motion  Axial flow by combining rocking motion with the time-dependent rotational speed of a rotating bottle
The method of enhancing mixing of a rotary bottle that introduces controlled disturbances at about 0 to about 50 rpm
Rotation speed, rotation speed change frequency of about 0 to about 1 per rotation, about 0 ° to about +10 degrees
Or a swing angle of -10 degrees and a frequency of swing versus rotation of about 0 to about 31.4.
Stipulated.

[0020]Time-dependent rotation direction of rotating bottle  Initially, the rotating bottle was rotated back and forth. Intuitively, when the direction of flow changes, particles
Was expected to continue descending in the fluid. Using a preliminary model of the two-dimensional flow
The numerical results used confirm this prediction in practice, but sedimentation enhancement depends on the sedimentation velocity.
Variability.

Although not calculated in this case, it is possible to estimate the cell sedimentation rate experimentally. Cell sedimentation in a stationary container was directly observable with a stereoscope at 100x magnification connected to a video camera and VCR. Next, the sedimentation velocity was estimated by slowly reproducing the cell movements recorded during sedimentation in the visual field.

[0022]Time-dependent rotation direction of a rotating bottle with oscillating motion  Axial flow by combining time-dependent rotation direction and oscillating motion of a rotating bottle
The method of mixing the rotary bottles to introduce a controlled disturbance to
Rotational speed (number of rotations), frequency of reversal of rotation direction from about 0 to about 1 per rotation, from about 0 ° to about
+10 degree or −10 degree swing angle and about 0 to about 31.4 swing versus rotation frequency
Specified by degrees.

[0023]Time-dependent rotation speed of a rotating bottle combining time-dependent rotation direction and rocking motion  Combining the time-dependent rotation direction and the oscillating motion and time-dependent rotation speed of a rotating bottle
The method of mixing rotary bottles, thereby introducing a controlled disturbance to the axial flow, further comprises:
A rotation speed of about 0 to about 50 rpm, a rotation direction reversal frequency of about 0 to about 1 per rotation,
A swing angle of about 0 ° to about +10 degrees or −10 degrees, and a swing angle of about 0 to about 31.4
It is defined by the frequency of dynamic pair rotation.

[0024] Typical parameters and preferred values are as follows: rotation speed is 0-50 rpm
, Preferably 0.1-5 rpm; the swing angle is 0- ± 20 degrees, preferably 0- ± 1.
0 degree; the magnitude of rotation speed change is 0% to 100% of low speed to high speed, and a preferable value is 50% or less of constant speed to high speed; the rotation speed change frequency is 0 to 10 times the rotation speed, preferably the rotation speed. 0.05-0.5 times; rotation direction reversal frequency is 0-2 times, preferably 0.5 times the rotation speed.

The present invention will be more fully understood by the following non-limiting examples.

Embodiment 1Development and experimental validation of computational models of fluid and cell motion in rotating bottles  Flow field solution Complete the standard rotary bottle using the commercial CFD software package FLUENT.
The whole three-dimensional flow field was obtained. FLUENT and similar software packages
Ever used by several researchers to solve fluid dynamics problems
[A. K. Majumdar, R .; H. Whitesides, S.M. L.
Jenkins, and D.J. L. Bacchus, J .; Prop. 6,5 (1
990); Dupont, M .; Pourkashanian, A .; Will
iams, and R.S. Wooley, Fuel 72, 497 (1993);
J. Seth and W.C. R. Wilcox, J .; Crystal Grow
th 114: 357 (1991); Y. Tang, J. et al. J. Ou, R .; H
. Heist, S.M. H. Chen and A. J. Dukat, Ind En
g. Chem. Res. 32, 1727 (1993); D. Swanson
, F. J. Muzzio, A .; Annapragada, and A.A. Adje
i, Int. J. Pharm 142, 33 (1996); M. Ho
bbs and F.S. J. See Muzzio, Chem. Eng. Sci. (199
It will be published in 7 years). ].

The equation of motion for the flow is the well-known Navier-Stokes equation, which includes the two-component momentum equation and the continuity equation for a two-dimensional flow;

[0028]

(Equation 1) [Where u is dimensionless velocity, p is dimensionless pressure, Re in equation (1) is Reynolds number, defined as Re = ρvL / μ, ρ and μ are density and viscosity of fluid, v and L represents a linear rotation speed and the height of the liquid in the bottle, respectively, Fr is the Froude number, defined as Fr = gL / v ^2, g represents a gravity]. The reason for choosing the liquid height as a measure of the natural length of the stream is that the liquid height is a measure of the diffusion length of the system. Viscous forces are observed as diffusive movement of momentum, and kinematic viscosity (μ / ρ) plays a role in momentum diffusivity. Thus, the depth of the liquid is a natural length criterion for the action of viscous forces.

The experimental geometry is shown in FIG. To obtain a velocity field, (a) the flow is stationary, (b) the flow is Stokes' regime (state of creep flow), (c) the free boundary is flat and horizontal (boundary Ignoring the surface tension and viscous drag at). Under the conditions used in the biotechnology industry, Re =
It is confirmed that the assumption of the creep flow is appropriate.

The physical domain was segmented using a curved mesh. The length-to-diameter ratio of the bottle is 2: 1 because of the symmetry of the rotating bottle, but a length-to-diameter ratio of 1: 1 was used because only one half of the system needed to be simulated. The height of the liquid in the bottle is 1 /
3, which is consistent with the industrial situation. A calculation grid with a structure of 80 × 20 × 94 is used. FIG. 1b shows a schematic of the z-plane grid (higher node densities were used for the calculations). By creating multiple meshes with different node densities, the permissible node density of the computational mesh was found. The number of mesh nodes was sequentially increased by 25%, and the process was repeated until the average speed difference between two successive meshes became less than 3%. When the average velocity difference was less than 3%, a calculated mesh was made using the lower mesh density.

The velocity and pressure fields were obtained by iteration using a Sun SPARC 20 workstation. The convergence of the solution required about 140 Mb of RAM and 60 hours of CPU time. A reference value of 10 ^-6 was used for each of the normalized velocity and pressure residuals to determine convergence. Solver for each iteration
Was normalized to the residual values obtained after the second iteration. To test the validity of the convergence criterion value, a simulation was performed using a convergence criterion value in the range of 10 ^-5 -10 ^-6 . When the convergence criterion value changed within this range, there was no significant change in the velocity field (the change in velocity amount was <1.0% on average). Sensitivity analysis (ie, orbit closure) has shown that such meshes are sufficiently accurate. This flow data was used for all subsequent particle tracking simulations.

The conditions adopted for the simulation of the basic case are Ω = 0.25 rpm and Reynolds number Re = 0.3, where Re = ρvD / μ, v is the linear rotation speed, ρ is the fluid density, and μ is the fluid Is the viscosity and the height of the liquid is the characteristic diameter D. When Re is this value, the flow can be considered to be creep flow law. Assuming that the flow was slow enough, it can be assumed that the free surface was always horizontal and there was no surface tension effect on the bottle wall. Accordingly, the free surface was modeled as an impermeable, frictionless wall (ie, a surface that did not exert any stress on the fluid). The particles in the stream are sufficiently dilute that they do not interact with each other or have no effect on the fluid flow. Assuming the particles were large enough, Brown's force was ignored. All the assumptions used here are valid for most rotary bottle industrial uses.

Fluid Velocity Field in a Rotating Bottle Two flow visualization experiments were performed to verify the simulation. For the experiment, a glass rotary bottle having a diameter of 10 cm and a length of 20 cm was used. Glycerin having a density of 1.26 g / cm ³ and a viscosity of 1.25 Pas was used as a working fluid. The bottle was rotated using a device consisting of a set of rollers whose speed and direction of rotation could be precisely controlled by a computer. The rotation speed used for the two experiments was 0.25,
According to this, Re was 0.04, which was sufficiently within the range of the creep flow law. First, the velocity field was verified by comparison with PIV results. The use of particle imaging techniques to study fluid flow problems is well described in the literature and is described in R.S. J. Adrian, Ann. Rev .. Fluid Mech. 23
, 261 (1991). The text provides information about our particular system. 12 μm diameter silver-coated hollow glass spheres with a specific gravity of 1.17 (Potter's Industries) were sprinkled into the stream. 10mJ
A New Wave Research pulsed miniature YAG laser was used as the irradiation source. The laser beam passed through a series of optics to produce a 1 mm thick light sheet (focal length: 1 m). A 32 × 32 grid was analyzed using cross-correlation data collection with 10,000 μs between laser pulses. From the PIV, data in the form of the direction and absolute value of the velocity at the center of each inquiry area were obtained.

The experimental velocity field was measured in a vertical plane in the center of the bottle. 2a and 2b, respectively.
3 shows velocity vector fields from experiments (2a) and calculations (2b). The length and color of the arrow represent the absolute value of the velocity in the plane from low (dark blue) to high (red). As can be seen in these figures, both velocity fields are qualitatively equal, except that in the case of the experiment there is some small scattering near the free surface of the liquid.

The computational velocity field was further verified by comparing the mixing patterns obtained with the passive tracer on the center plane of the bottle in the case of calculations and in the case of experiments. The first step in the simulation of this mixing pattern is to determine the trajectory followed by the point particle as a result of the equation of motion dx / dt = v (3) where x is the position of the particle and v is the fluid velocity. FIG.
Figure 3 shows the predicted flow path of the flowing particles using the fourth-order Runge-Kutta integral of equation (3). This flow is also streamlined since the flow is steady and the particles passively follow the flow. The stagnation point is on the line of symmetry below the free surface by 0.6 unit length (the unit length is the height of the liquid). A similar method was used to simulate the evolution of a vertical filament initially placed in the center of the bottle (FIG. 4a). A line consisting of 10,000 particles moving at the speed of the flow was constructed. 4b-4e show the shape of the line as determined from the particle position when the bottle has been rotated 4, 8, 16 and 32 times. The line is twisted around the stagnation point as the particles are moved by the flow. The line extends linearly within this steady two-dimensional flow. In order to confirm the simulation, the development of the fluid line was observed experimentally. FIG. 5 shows an initial state composed of vertical lines of the coloring fluid arranged on the center plane of the rotating bottle, and FIG. 6 shows the development of the dye lines after 20 rotations. Again,
Excellent qualitative agreement between experiment and simulation was obtained. Therefore, it was concluded that the simulation actually represented accurately the velocity field in the rotating bottle.

After validating the calculated velocity field, it was used to obtain a detailed characterization of the fluid motion in the bottle. Clearly illustrating the three-dimensional velocity vector field is not an easy task. In the present application, a two-dimensional diagram (c) of a velocity field expressing a component in a plane by a vector
ut), the overall size of the velocity field is indicated by a colored contour where red means fast and blue means slow.

FIG. 7 shows a vertical cross section of the bottle at 2 cm intervals (ie, z = 0.0 is 0.0 mm from the end wall).
m, and z = 0.1 m indicates the center of symmetry of the rotating bottle). FIG. 7f corresponds to the center of the bottle and is therefore equivalent to FIG. 2b. 7d and 7e are also equal to FIG. 7f,
It shows that the end effect has little or no effect on the fluid flow 5 cm from the end of the bottle. The velocity field has mirror symmetry about the mid-plane (predicted from the symmetry of the boundary) and bilateral asymmetry (predicted from creep flow conditions and rotational symmetry of the bottle). This observation has important implications, as described below, since the flow induced by the bottle end is the only means of axial stirring.

FIG. 7 a represents the velocity field at the end of the rotating bottle. Because the bottle wall is non-slip, all fluid flows counterclockwise along the bottle wall. When the fluid reaches the free surface (on the right), it flows outward in the z-direction away from the wall. On the left, in the immediate vicinity of the wall, the fluid is pulled downward. This movement is "enhanced" by an inward flow from the z-direction. 7b and 7c show the presence of a fast component near the liquid surface caused by the end effect. However, a detailed examination of the velocity vectors shown in FIGS.
This shows that the x and y components of the velocity field are hardly affected by the end effect. Such an effect mainly appears in the axial (z) component of the velocity field.

The end wall effect is more clearly shown by the plan view of the bottle. FIG. 8 shows a contour plot of the velocity just below the liquid free surface. The bottle edge is at the bottom of this figure and the plane of symmetry is at the top. In this figure, the flow in the axis (z) direction is clearly observed. Fluid flows upward from the lower right corner of the end wall, travels in a counterclockwise loop, then flows downward, and returns to the lower right corner by the end wall flow. There is a stagnation point 0.46 cm away from the edge of the bottle, as shown in dark blue. The effect of the edge effect is less on the flow moving towards the center of the bottle. The velocity field difference between the 2-D symmetry plane and the plane 5 cm away from the end wall is 2%
There is only. The surface flow in this region is from right to left.

Alternatively, calculating a flow path provides a diagram of a three-dimensional flow pattern in a rotating bottle. FIG. 9 shows six lines of fluid particles initially placed 0.2 cm away from the bottle bottom. The center of the bottle is on the left and the end wall is on the right. Looking at the line from left to right, you can observe the effect of the end wall on the flow field. The middle plane line is a two-dimensional vertical loop equal to the line observed in FIG. As it moves toward the end of the bottle, the line begins to turn towards the center of the bottle near the free surface. All the illustrated routes are closed loops. The route on the center plane of the bottle is a two-dimensional loop,
Lines away from the plane of symmetry are three-dimensional loops. However, in both situations, the subsequent results are the same. Fluid particles in the stream are trapped in closed periodic trajectories and do not contact other parts of the stream. Such a regular pattern of motion indicates that the mechanical agitation of the fluid in this stream is very poor and homogenization occurs primarily by diffusion. Experimental proof of this observation and ways to overcome the limitations of mixing are discussed below.

Movement of Cells in a Rotating Bottle Maxey and Riley [M. R. Maxey and J.M. J. R
The formula proposed by iley, Fluids 26, 883 (1983.):

[0042]

(Equation 2) (In the formula,

[0043]

(Equation 3) Is used as a starting point to model finite small particles in the flow field.

In these equations, u is the fluid velocity vector, v is the cell velocity vector, a is the cell radius, v is the kinematic viscosity of the fluid, m _p and m _F are the cell weight and the fluid displaced by the cell, respectively. Corresponding to the weight of The five terms on the right-hand side of the equation correspond to buoyancy, pressure, flow history effects, added mass effects, and Stokes drag. This equation can be considerably simplified to the problem of the rotating bottle. Since u is known from the numerical solution of the flow, the relative magnitude of each term can be calculated. Analysis shows that the key terms are inertia, buoyancy and drag, and therefore the equation:

[0045]

(Equation 4) Where G = 2 (γ-1) gL / [2γ + 1) U ² ] and S _tk = (2γ + 1) a ² (9 vL); γ is the particle density relative to the fluid density, L is the depth of the fluid, and U is The rotation speed of the bottle]. This equation can be further simplified assuming that the particles are cells and that the rotational speed and liquid are those typically used in the biotechnology industry. Typical examples are G〜O (10 ⁺³ ) and S _tk ＯO (10 ^-6 ).

This greatly simplifies the problem because the inertia term can be ignored. The particle velocity is given by the formula:

[0047]

(Equation 5) Where v is the total particle velocity and is simply the sum of the fluid velocity u and the final settling velocity Vs of the particle. Once v is known, the position of the particles is determined using the standard fourth-order Runge-Kutta method using the equation

[0048]

(Equation 6) Can be found by numerical integration of. To understand the behavior of small particles in a rotating bottle with a steady rotation, equations (6) and (7) are used to simulate the motion of particles on the center plane of the bottle. FIG. 10 shows the trajectory of a particle with a sedimentation velocity Vs = 0.05 U, where U is the dimensionless tangential velocity of the bottle wall. Six particles were initially placed on the vertical center line. The particles behaved differently. The two particles closest to the free surface reached the rotating wall within one revolution of the bottle, while the remaining four particles were moving in a closed orbit. Such particles never reach the bottom wall. This simulation shows that whether particles settle or not depends not only on the sedimentation velocity but also on the initial position in the flow field. Further, since the sedimentation velocity used in this simulation is much greater than the sedimentation velocity corresponding to a real system, FIG. 10 actually shows that a significant portion of the cells in the spinning bottle are actually recirculating inside the underflow. Indicates that it will be caught in orbit indefinitely and will never reach the bottle wall.

The results of this simulation were experimentally studied using a long-exposure photography method that detects a route followed by fluorescent particles having a sedimentation velocity similar to the sedimentation velocity corresponding to FIG. 200 μm fluorescent polystyrene particles were placed on the center surface of the rotary bottle. The particles had a final sedimentation rate of about 0.5 cm / min or 0.06 U, where U is the linear rotation speed of the tumbling bottle. The path of the particles during one revolution of the bottle was photographed under fluorescence using long exposure photography. FIG. 11 shows the results obtained in such an experiment. The initial position of the particles was about 0.6 unit length from the free surface (the unit length is the height of the fluid)
. The route shown in FIG. 11 is very similar to the pattern shown in FIG. 10, which is a simulation of particles of substantially similar settling velocity. When the particles were placed in another initial position, closer to the center of the bottle, the particles followed another closed path. When placed close enough to the top surface, the particles settled and attached to the bottle wall.

Detailed Analysis of Cell Sedimentation: Particle Sedimentation Results Tracking a large number of cells over time provides a more complete picture of cell behavior. To perform this simulation, 20,000 particles were placed in a uniform distribution throughout the central cross section of the bottle and these particles were tracked during 20 rotations of the bottle. When one particle touched the bottle wall, the particle was (optionally) assumed to have "settled" and was removed from the simulation. FIG. 12 shows that the sedimentation velocities Vs = 0, 0. The final positions of the particles obtained from such simulations of 02U, 0.05U, 0.1U and 0.2U are shown. For these simulations, the bottle is U =
Rotated counterclockwise at a dimensionless linear speed of 1.0. In the case of Vs = 0 (FIG. 12), such particles are actually fluid particles, so the structure of the particle positions is equal to the structure of the streamlines of the flow. The position of the particles forms a closed orbit, indicating that all particles are confined inside the closed orbit. As expected, no settling occurs in the stream. As Vs increases, the course of the particles is disturbed and becomes non-streamlined, but when Vs = 0.02 U, as shown in FIG. 12b, most of the particles are trapped in closed periodic orbits. And never reach the wall. Only particles that were initially very close to the free surface and the bottle wall can reach the bottle boundary. As the sedimentation velocity increases, more particles settle, but most of the particles can reach the bottle wall only when the value of Vs is large (ie, Vs = 0.2 U, Figure 12e).

Such a simulation gives information about the dynamic behavior of the particle motion,
It is not possible to estimate the sedimentation velocity from such a simulation. To obtain the spatial distribution of particle settling time, the settling time of each particle was recorded and plotted as a function of the initial position of the particles. The sedimentation time distribution inside the bottle is shown in FIG. Rotate the bottle clockwise at the dimensionless linear velocity U = 1.0 at the wall. The results were Vs = 0.02 (FIG. 13a), 0.05 (FIG. 13b), 0.1 (FIG. 13c) and 0.2 (FIG. 13c).
Corresponds to d). Colors from dark blue to red represent the residence time of the particles from 0 to the maximum calculation time. The solid core region of the red region represents particles that do not settle at all.
At a low value of Vs = 0.02 (FIG. 13a), only a narrow band of particles near the right bottom of the bottle below the upper free surface settles. The width of this band increases with increasing Vs and the size of the red core shrinks. The tip of this core region is always in the upper left corner of the stream and is inclined toward the bottom left of the bottle. If Vs = 0.1 (Figure 13b), particles in most areas of the bottle will settle. As Vs increases to 0.2 (FIG. 13c), almost all of the particles settle on the wall. The spatial distribution of settling times of the particles in the stream is clearly indicated by shading. In this flow direction, short-lived particles are always on the right side of the bottle. When the flow direction is reversed, the system is symmetric,
Will appear as a mirror image of itself. These results clearly show that the distribution of particle residence time is strongly affected by stream recirculation.

FIG. 14 shows the particle settling velocity. In this figure, the number of particles suspended in the stream at a given time is plotted against time. From this figure, it is observed that the entire process of sedimentation is non-linear (probably except when Vs = 0.2). The particles settle on the wall relatively quickly in the early stages, and then the sedimentation rate (slope of the graph) levels out in a relatively short time of about 8-12 unit hours. If the sedimentation velocity is high (Vs = 0.2, test for the heaviest particles), the particles sediment at a nearly constant velocity.

Example 2Enhancement of cell deposition by rotational disturbance  Use of Unsteady Rotation to Enhance Sedimentation A comparison of FIGS. 3 and 12 shows that the non-sedimentation
Indicates that motion is induced. In the two-dimensional flow field diagram, the flow behavior is multiple nested
Shown as a closed loop. One particle is in the lower part of the flow (below the stagnation point)
When present, the particles descend in the loop toward the outer loop. However,
If the particles do not reach the wall, they will then move to the upper part of the flow and then
Descend toward the hoop. Due to the combination of these two effects, the particles will have a closed orbit
Move up and down within. In fact, even without Brownian motion, particles have two phenomena:
It may indicate either sinking to a wall or moving in a closed trajectory.

By forcing the flow unsteadily, such closed orbits can be broken, and consequently, sedimentation can be enhanced. This hypothesis was tested by disrupting the flow. One way to reach this goal is to use a transient flow. The easiest and most controllable transient flow is a time-periodic flow. There are various ways to introduce periodicity into the flow. One way is to periodically reverse the direction of flow. The flow period can be defined as the total time of forward flow and backward flow. The transient effect that occurs when the flow reverses direction is usually large, but in the case of the creep flow law used in this example, this effect can be neglected because the inertia term of the Navier-Stokes equation can be neglected. . Accordingly, a periodic flow can be simulated by two equal steady flows, each being a mirror image of each other, acting alternately by a predetermined amount of time.

In the case of passive tracer mixing, such periodic flow would not be improved at all since the particles only move back and forth along the same streamline, but the sedimentation of the heavy particles is caused by a periodic reversal in the direction of rotation. Astonishingly enhanced by The sedimentation behavior of a particle is defined as the complete elimination of the bottom wall between a pair of inversions (ie, the flow period), measured in dimensionless units, where one unit is equal to 液体 the depth of the liquid. It greatly depends on the value of T. For example, if the bottle completes one revolution during this inversion, a value of T = 37.7 is obtained. For some values of T, complete sedimentation is achieved in a short time, even at much slower sedimentation rates than discussed above for steady flow.

The rate of particle settling in a periodic stream was tested by evenly distributing 6,540 particles in the flow domain. As mentioned above, particles impacting the wall or free surface were considered settled on the wall. Two small sedimentation velocity values, Vs = 0.002 and 0. 005 were tested. In each case the particles were tracked for 30 cycles and the number of particles suspended in the stream was plotted against time. FIG. 15 shows the result when Vs = 0.005. From this figure, it is observed that the sedimentation process is linear for all values of T. When T is a small value, that is, when T <25, almost all particles settle very quickly,
The smaller the T, the faster the particles settle. However, at T = 25, almost no sedimentation occurs. The same applies to the case of T = 50. The particle settling velocity increases from a small value of T to T = 25 and then decreases to T = 35. For T = 35 and 40, the sedimentation velocity is almost the same during most of the flow process. However, at a later stage, the sedimentation at T = 40 continues at a linear velocity, but the sedimentation at T = 35 slows down. Thus, the fastest sedimentation is achieved when T is small (T <25) or between T = 35 and T = 40. Of these two conditions, the latter is preferred. The reason is that for T <25, some regions of the bottle wall do not come into contact with the liquid because the bottle does not complete one revolution between inversions. Such conditions lead to starvation and death of cells in such areas of the bottle wall, thus reducing processing efficiency.

Example 3Use of rocking motion to enhance axial mixing  As described above, the flow pattern solved by simulation is
Directive suggests slow mechanical mixing of fluids. This prediction is based on the pH indicator (bromothiocyanate).
(Mole Blue) and acid-base neutralization to confirm the mixing behavior.
[D. J. Lamberto, F .; J. Muzzio, and A.M. L. T
onovich, Chem. Eng. Sci. 51: 5,733 (1996)
. ]. From a set of rollers that can precisely control the speed and direction of rotation by computer
An experiment was performed with the apparatus (FIG. 20). To obtain a creep law flow,
Serine was used as the working fluid. First add glycerin to the indicator and 0.5 ml
Mix with 1M NaOH. Glycerin turned deep blue upon mixing. apparatus
, Put about 20 ml of glycerin from the bottle with a syringe,
Upon mixing with 1 M HCl, the fluid turned yellow. This fluid is then rotated
Refill with a syringe near the back wall of the bottle (bottom of the bottle when the bottle is upright), and specially modified
The spatula was carefully mixed with the surrounding fluid at the bottom of the bottle.

After re-injecting the acidic glycerin into the rotary bottle, the mixer was activated and the bottle was rotated at a constant speed. Figures 21a-c show the results of this experiment. The initial conditions are shown in FIG.
The mixing after 32 rotations is shown in FIG. 21b, and the mixing after 64 rotations is shown in FIG. 21c. These figures clearly show the effect of the symmetry plane present in the center of the bottle. After 32 rotations, mixing in each of the bottle halves is complete. This mixing is caused by the axial movement of the fluid due to the end wall effect. However, the mixing between the two halves of the bottle is still minimal after 64 rotations of the bottle. Neutralization of the base on the right side occurs only by diffusion.

Recent experiments dealing with the mixing of dry powder in a rotating and oscillating cylinder have shown that small vertical oscillating motions can significantly enhance axial oscillating motion [C . Wrightman, P .; R. Mort, E .; K. Gleason a
nd F. J. Muzzio, Powder Tech. 84: 3,231 (1
995). ]. In the present invention, this method was implemented with the aim of enhancing mixing in the rotating bottle. (A) mixing using a rocking frequency of 1.6 rotations of the bottle per rocking cycle, and (b) mixing using a rocking frequency of 3.2 rotations of the bottle per rocking cycle,
Were tested.

FIGS. 21 d-f show the effect of rocking on mixing of the system. 1 bottle per swing.
The rotary bottle was rocked about 12 degrees at a rocking speed of 6 rotations. It can be observed that the mixing of the system is substantially improved as a result of the rocking. Similarly, Figures 21g-j show mixing with a rocking speed of 3.2 rotations of the bottle per rocking. These results demonstrate that rocking actually enhances mixing, and that mixing is nearly complete after 64 revolutions. Furthermore,
Since the wobble disrupts the aforementioned recirculating flow pattern, it is believed that the circular motion of the cells is also disrupted, promoting sedimentation. However, it is important to recognize whether rocking should be used to enhance the mixing of the cell growth process. The frequency and amplitude of the rocking must be selected taking into account that the cells need to be immersed in the nutrient medium as uniformly as possible. If an integer number of revolutions per rocking cycle is selected, the cells at the top of the bottle will always be the same when the rocking reaches the maximum tilt angle, and will be substantially shorter in nutrients than the cells at the bottom. Will not be able to contact. The reason for choosing the rocking frequencies of 1.6 and 3.2 rotations per rocking cycle is that at these frequencies cells are less likely to come in contact with nutrients closer to the mouth of the bottle, This is because the cells can contact the nutrients fairly evenly.

Embodiment 4Effect of bottle rotation speed on cell culture experiments: time-dependent rotation speed  Thaw frozen MRC-5 cells and place cells in T-culture bottles or flat stationary growth surfaces
The cells were cultured in a certain Nune Cell Factories. Cells are grown in vegetative culture
They adhered to the bottom of these vessels immersed in the ground and proliferated. Trypsin treatment every 6 days
Cells were harvested from the surface by enzymatic means known in the art. Then you got
The cell suspension was diluted and placed in multiple containers for further growth.

On day 27 of treatment, the freshly trypsinized and concentrated cell suspension was introduced into a rotary bottle with 125 mL of growth medium. The bottle was spun at 0.25 or 0.5 rpm and incubated at 37 ° C. On day 33, an additional 300 mL of growth medium was added to the bottle. As shown below, the cell yield increased at higher rotation speeds. The numbers represent the average number of cells per bottle produced on day 35 of treatment.
The number of cells is increased by 23% ± 10% compared to the rotation speed of 0.25 rpm.

[0063]

[Table 1]

Prolonging the exponential growth phase further increases cell yield. See FIG. 18, which shows the daily growth curves for two of the five tests. FIG. 18 shows that the increase in cell number was not due to an increase in growth rate but to an extension of the exponential growth phase of the culture. This may be due to the improved transport of nutrients, gases and waste products due to the faster rotation.

Example 5 Results of RPM Changes in Productivity Enhancement The spinning bottle cultures of Tests 4 and 5 above were then infected by addition to freshly trypsinized virus-infected MRC-5 "working seed" cells. When the virus-infected cells were added to the liquid nutrient medium in the jar, the cells attached to the uninfected cell monolayer established on the inner surface of the jar. After adding the "working seeds" to the jar, the jar was rotated at an alternating speed.

The following table shows the results normalized from the experiments.

[0067]

[Table 2]

For each of the two experiments, each of the four experimental groups was tested in five duplicate plaque assays to assess efficacy. The combined strategy of high speed rotation during cell growth and constant speed rotation during the initial infection period increased production by 32% with a 95% confidence interval of 15%, showing a statistically significant result.

As described above, the spinning bottle produces an annular liquid stream into which varicella-infected cells can be "captured". These infected cells gradually lose their infectivity as they circulate in the liquid phase (half-life of the infectivity is less than 5 hours). Reducing the rotation speed of the bottle does not disrupt the annular flow, but modeling has shown that more captured cells can reach the surface more quickly by sedimentation, as confirmed in these experiments. .

Transport of Virus-Infected Cells to Rotary Bottle Surface FIGS. 15-17 show that reducing the rotational speed accelerates transport of infected cells to the cell culture established on the inner surface of the rotary bottle. . Minimizing the time that infected cells stay in the culture supernatant is important for two reasons. First, the residence time in the supernatant is the processing time that does not produce a product because a given viral infectious particle does not infect the cell monolayer. Second, the half-life of virus activity is less than 5 hours. The residence time in the supernatant is directly manifested as a breakdown of the viral material.

FIGS. 15 and 17 show the number of infected cells present in the supernatant at several different rotation speeds as a function of time. FIG. 15 shows that at a high rotation speed of １ ／ rpm, more infected cells remain in the supernatant than the control at the normal rotation speed (１ ／ rpm) and the T-type culture bottle stationary control. FIG. 16 shows that the disappearance of infected cells in the supernatant correlates with the appearance of infected foci in the cell monolayer. In all cases, the faster the rpm, the smaller the number of foci per unit area.

FIG. 17 shows similar data obtained from experiments tested at １ ／ and ８ rpm. Also in this case, the lower the rpm, the more the infected cells are transported to the surface. The technique used for cell counting in FIG. 17 was much more sensitive than the technique used for cell counting in FIG. Thus, the two experiments gave different results for the 1/4 rpm control.

Example 6 Materials and Methods Varicella Oka strain was obtained from Biken Institute, Japan.
MRC-5 cells were obtained from the ATCC or NIBSC and purchased from Merck at Manu.
It was used for making factor's Working Cell Bank (MWCB). MEM is manufactured by Merck and contains fetal bovine serum (FBS)
Neomycin and glutamine were supplemented. HBME was purchased from GIBCO.
Horseradish peroxidase-conjugated donkey anti-goat antibody (affinity purified F (a
b ′) 2 fragment) is Jackson Immunoresearch L
abs, Inc. , West Grove, PA, (Cat # 705-036-
147). Goat anti-varicella antiserum is available from Lampire, Inc. (Lot
2200). 3,3-Diaminobenzidine (DAB) is Sigma
a, prepared as a 25 mg / ml aqueous solution and stored at -20 ° C. Just before use, thaw the DAB pre-prepared solution and use a peroxide / nickel enhancement solution (Amersham).
) And diluted in phosphate buffer.

Establishment of Cell Monolayers Stationary Cell Swelling : MRC-5 ampoules (ATCC No. CCL171) are thawed and cells are plated in T-type culture bottles or Nunc Cell Factory, a flat stationary growth surface.
s. Cells were added to BME medium supplemented with 10% FBS, 50 μg / mL neomycin and 2 mM glutamine and incubated at 37 ° C.
Cells were harvested from the surface by enzymatic means using trypsin every 3-9 days (depending on the actual method). The resulting cell suspension was diluted and placed in more containers for further growth.

Roller Cell Expansion : Freshly trypsinized concentrated cell suspension was placed in a rotary bottle with MEM growth medium supplemented with 10% FBS, 50 μg / mL neomycin and 2 mM glutamine. The bottle was incubated at 37 ° C. and spun at the desired speed. After 4-6 days (depending on the method used), the jar was re-fed with fresh nutrient medium.

Cell Sedimentation Analysis Rotary Bottle Infection : Rotor bottle cultures with established cell monolayers on the roller bottle surface were infected with freshly trypsinized virus-infected MRC-5 cells. The virus-infected cells were added to the liquid nutrient medium in the rotating bottle 6-8 days after the cells were introduced into the starting rotating bottle.

Sonication of Harvested Fluid Samples : The samples were thawed in water at 20-30 ° C. with frequent stirring. The sonicator was calibrated so that the tip of the sonicator was immersed in 20 mL of room temperature water and excited for 1 minute to raise the water temperature by 5 ° C. Next, the tip of the sonicator was sterilized by applying 90% isopropyl alcohol. Each of the thawed samples was sonicated for 1 minute in 20 mL aliquots. The sonicated samples were then frozen at -70 ° C and used for titer testing.

Titer Test : The number of plaque forming units (PFU) per mL of frozen sample of the harvest was tested using a standard plaque assay.

Varicella plaque assay : MRC-5 cells were grown on 60 mm plates using 5 mL per plate of BME containing 10% FBS, 0.2% glutamine and 0.05% neomycin. Ten plates were used per sample and five plates per dilution. The plates were then incubated for 2 days at 35.5 ° C. in a 3% CO ₂ gas environment. Remove the cell growth medium before adding the sample and add 2%
MEM containing FBS, 0.2% glutamine and 0.05% neomycin was added to each plate in a volume of 5 ml per plate. 0.1 ml of diluted virus sample was added to each plate. Samples were diluted to ensure that plaques in the range of 15-80 plaques per plate could be counted. Plates were mixed well and incubated for 7 days at 35.5 ° C. in a 3% CO ₂ gas environment. After incubation, the medium is removed by aspiration and the medium containing 1% v / v glacial acetic acid is removed.
2 ml of the dye containing 0.2% Coomassie Blue in 0% ethanol was added to each plate. After 15 minutes at room temperature, the dye was removed and the plate was washed with cold water. The plate was inverted and dried. The plaques on each plate were then counted using a light box to help visualize the plates.

Particle Size Analysis : Particle counting and particle size analysis were performed using a 48 μm diameter Elzone particle size analyzer model. The sample was diluted to 20 ml with PBS. The diluted sample was placed on the sample holder of an analyzer calibrated with particles of known diameter (10 and 20 μm microspheres).

In situ immunostaining procedure : The medium was aspirated from the culture vessels (T-type or rotary culture bottles) using vacuum. Next, the culture vessel was washed three times with PBS supplemented with 1% BSA. After a final wash with PBS, a fixer consisting of 5% acetic acid, 5% water and 90% methanol was added to the culture vessel and allowed to contact the entire surface for 15-30 minutes. Next, methanol was sucked from the culture vessel using vacuum. Then, the culture vessel was washed three more times with PBS.
Stored at 4-8 ° C. until overlaying and staining the BS.

The fixed bottles were immunostained by the two-antibody method. First, 2-3 ml of goat anti-varicella antiserum diluted 1/500 in PBS containing 1% BSA was added to the culture vessel, and the entire growth surface of the T-type culture bottle and 3-4 cm along one side of the rotating bottle were added. The surface of the width zone was coated. Next, the container was placed on a rocking table, and rocked gently for 1-2 hours at room temperature to bind the first antibody. The culture vessel was washed three times with PBS to remove unbound antibody, and 3 ml of anti-goat antibody diluted 1/500 was added. The container was incubated again for 30-45 minutes at room temperature with gentle rocking. Next, it was washed three times with PBS to remove unbound antibody. Then, 10 ml of peroxidase substrate solution was added to each culture vessel and the vessels were incubated at 37 ° C. until infected cell foci appeared. Next, the peroxidase solution was aspirated from the container using vacuum and the container was again washed three times with PBS. The stained area was kept under the PBS overlay and the containers were stored at 4-8 ° C. The infected foci were observed with a microscope at a magnification of 10 times.

[Brief description of the drawings]

This patent application contains at least one color drawing. Copies of the color drawing (s) may be obtained by applying to the Patent Office for a fee.

FIG. 1A is a schematic diagram of a rotating bottle, and FIG. 1B is a schematic diagram of a calculation grid used in a simulation.

FIG. 2A is a velocity field at the center plane of the bottle measured by particle imaging velocimetry;
FIG. 2B is a finite element result. Velocity field on the center plane of the bottle.

FIG. 3 shows a calculated streamline of the flow on the center plane of the bottle in a steady flow state.

FIG. 4 shows the development of a vertical line arranged in a flow field. 4A shows the initial state, and FIGS. 4B-4E show the lines after the bottle has rotated 4, 8, 16 and 32, respectively, from the initial state.

FIG. 5 is an initial state of a flow visualization experiment. The fluorescent dye was placed axially in the center of the bottle so that the edge effect was nullified.

FIG. 6 is an experimental verification of deployment of a vertical line arranged in a flow field.

FIG. 7 Finite element results. Velocity fields in multiple vertical planes spaced along the axis of the bottle.

FIG. 8 Finite element results. Horizontal velocity field near the top surface of the fluid.

FIG. 9: Passive tracer particle routes starting from various locations inside the bottle.

FIG. 10: Route of six particles arranged in the stream with sedimentation velocity Vs = 0.05 U.

FIG. 11: Experimental verification of the path of 0.06 U sedimentation velocity particles placed in a stream.

FIG. 12: Final positions of the particles corresponding to the sedimentation velocities Vs = 0.02U, 0.05U, 0.1U and 0.2U, respectively, of the particles evenly distributed in the central vertical section of the bottle at first.

FIG. 13: Initial values of particles corresponding to Vs = 0.02 U, (FIG. 13B) 0.05 U, (FIG. 13C) 0.1 U and (FIG. 13D) 0.2 U after 20 rotations of the bottle, respectively. Contour plot of particle residence time versus position. The range of dark blue to red colors in this figure corresponds to a residence time of 0 to infinity.

FIG. 14 shows the number of suspended particles in a stream at various settling speeds as a function of time.

FIG. 15 shows the percentage of cells remaining in the supernatant as a function of the rotation speed of the spinning bottle.

FIG. 16 shows the relative amount of foci on a cell monolayer as a function of rotation speed.

FIG. 17 shows the relative amount of foci on a cell monolayer at low rotation speed as a function of rotation speed.

FIG. 18 shows cell growth rate and final cell density as a function of spin rate.

FIG. 19 shows the number of suspended particles in a stream as a function of time at various rotation cycles. The same sedimentation velocity Vs = 0.003 U was used in all graphs.

FIG. 20: Experimental apparatus used to test mixing enhancement by introducing rocking motion.

FIG. 21a shows the effect of rocking on axial mixing. 21A-21C are 0, 32 and 6, respectively.
7 shows axial mixing without swinging after four rotations.

FIG. 21b: Effect of rocking on axial mixing. FIGS. 21D-21F show axial mixing after 0, 32, and 64 rotations, respectively, with 12.047 ° rocking and 1.6 rotations per rocking.

FIG. 21c: Effect of rocking on axial mixing. FIGS. 21G-21I show the axial mixing after 0, 32 and 64 rotations, respectively, with 12.047 ° rocking and 3.2 rotations per rocking.

FIG. 22 shows a roller holding machine capable of introducing a swinging motion.

FIG. 23A (FIG. 23A) Protrusions on the rotatable bottle and (FIG. 23B) for introducing a rocking movement provided on the rollers.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] October 25, 1999 (1999.10.25)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

────────────────────────────────────────────────── ─── Continued on the front page (31) Priority claim number 972718.2 (32) Priority date December 22, 1997 (December 22, 1997) (33) Priority claim country United Kingdom (GB) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM) , AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AU, AZ, BA, BB, BG, BR, BY, CA, CN, CU, CZ, EE, GD, GE , HR, HU, ID, IL, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LV, MD, MG, MK, MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, SL, TJ, TM, TR, TT, UA, US, UZ, VN, YU (71) Applicant Rutgers, The State University of New Jersey New Jersey, United States of America 08903, New Brunswick, Somerset Street, Old Queens (no address) (72) Inventor Bramble, Gijoy El, United States, New Jersey 07065, Lowway, East Lincoln Avenue 126 (72) Inventor Sears, The Ames A United States, New Jersey 07065, Lowway, East Linker Avenue 126 (72) Inventors Muttuio, Fernando Jei United States, New Jersey 08903, New Brunswick, Somerset Street, Old Queens (no address) F-term (reference) 4B065 BC08 BC18 BC26 CA44

Claims (29)

[Claims] 1. The method of claim 1, further comprising introducing controlled axial flow disturbances, cross-direction flow disturbances, or both axial and cross-direction flow disturbances into the rotational mixing in the rotary bottle. Mixed enhancement method. 2. The method according to claim 1, wherein the controlled axial flow disturbance is introduced by a combination of a vertical oscillating motion and a constant rotation of the rotatable bottle along a horizontal axis. A method for enhancing mixing of materials in a rotating bottle. 3. The method according to claim 2, wherein the oscillating movement is caused by oscillating rollers that drive the rotation of the rotatable bottle. 4. The oscillating motion is defined by an oscillating angle from about 0 degrees to about +10 degrees or -10 degrees and a frequency of about 0 to about 31.4 oscillating versus rotating. The method for enhancing the mixing of materials in a rotary bottle according to claim 3. 5. The method according to claim 2, wherein the oscillating motion is caused by providing a plurality of protrusions on a roller for driving the rotation of the rotatable bottle. 6. The method according to claim 2, wherein the oscillating movement is caused by providing a plurality of projections on the rotatable bottle. 7. The method of claim 1, wherein the controlled cross-flow disturbance is introduced by using a time-dependent rotational speed. 8. The method according to claim 7, wherein the rotation speed varies in a range of about 0.01 to about 10 times the rotation number of the bottle. 9. The method according to claim 7, wherein the rotation speed changes continuously or discontinuously with a time-dependent frequency. 10. The method of claim 1, wherein the controlled cross flow disturbance is introduced by reversing the direction of rotation of the bottle. 11. The method according to claim 10, wherein the direction of rotation changes in the range of about 0.01 to about 3 times the number of rotations of the rotary bottle. 12. The method according to claim 10, wherein the direction of rotation changes continuously or discontinuously with a time-dependent frequency. 13. The rotary bottle according to claim 1, wherein controlled axial and cross-flow disturbances are introduced by a combination of oscillating motion and time-dependent rotational speed. How to improve the mixing of materials. 14. The method according to claim 13, wherein the oscillating movement is caused by oscillating rollers that drive the rotation of the rotatable bottle. 15. The method according to claim 13, wherein the oscillating movement is caused by providing a projection on a roller for driving the rotation of the rotatable bottle. 16. The method according to claim 13, wherein the oscillating movement is caused by providing a projection on the rotatable bottle. 17. The oscillating motion is defined by an oscillating angle from about 0 degrees to about +10 degrees or -10 degrees and a frequency of oscillating versus rotation from about 0 to about 31.4, and is time dependent. 14. The method of claim 13, wherein the rotation speed changes at a frequency of about 0.01 to about 10 times the rotation speed of the jar. 18. The method according to claim 17, wherein the swing speed and the rotation speed change continuously or discontinuously with a time-dependent frequency. 19. The material in a rotary bottle according to claim 1, wherein the controlled disturbance of the axial and cross-flow is introduced by a combination of oscillating movement and time-dependent rotational speed. How to improve mixing. 20. The method according to claim 19, wherein the oscillating movement is caused by oscillating rollers driving the rotation of the rotatable bottle. 21. The method according to claim 19, wherein the oscillating movement is caused by providing a projection on a roller for driving the rotation of the rotatable bottle. 22. The method according to claim 19, wherein the oscillating movement is caused by providing a projection on the rotatable bottle. 23. The oscillating motion is defined by an oscillating angle from about 0 degrees to about +10 degrees or -10 degrees and a frequency of oscillating versus rotation from about 0 to about 31.4, and the time-dependent direction. 20. The method of claim 19, wherein f changes at a frequency of about 0.01 to about 3 times the number of revolutions of the jar. 24. The method according to claim 23, wherein the swing speed and the rotation direction change continuously or discontinuously with a time-dependent frequency. 25. The method according to claim 1, wherein the controlled disturbance of the axial and cross-directional flow is introduced by a combination of oscillating motion, time-dependent rotational speed and time-dependent rotational direction. Method of mixing and improving the materials in the rotating bottle. 26. The method according to claim 25, wherein the oscillating movement is caused by oscillating rollers driving the rotation of the rotatable bottle. 27. The method according to claim 25, wherein the oscillating movement is caused by providing a projection on a roller for driving the rotation of the rotatable bottle. 28. The method according to claim 25, wherein the oscillating movement is caused by providing a projection on the rotatable bottle. 29. The oscillating motion is defined by an oscillating angle from about 0 degrees to about +10 degrees or -10 degrees and a frequency of oscillating pair rotations from about 0 to about 31.4, and a time-dependent rotational direction. Wherein the rotation speed changes at a frequency of about 0.01 to about 3 times the rotation speed of the rotary bottle, and the rotation speed changes at a frequency of about 0.01 to about 10 times the rotation speed of the rotary bottle. 26. The method for enhancing mixing of materials in a rotary bottle according to 25.