WO2000029545A1

WO2000029545A1 - Method for performing cell culture

Info

Publication number: WO2000029545A1
Application number: PCT/GB1999/003768
Authority: WO
Inventors: Robert Cecil Brooks
Original assignee: Aea Technology Plc
Priority date: 1998-11-17
Filing date: 1999-11-11
Publication date: 2000-05-25
Also published as: GB9825054D0

Abstract

A suspension of cells in a liquid medium is enclosed in a vessel (12), the vessel comprising a plurality of equally-spaced chambers (24) forming a linear array, each chamber communicating with the next chamber in the array via a linking port (26) defined by a smoothly rounded wall portion. The chambers (24) may be of circular cross-section. The liquid medium may be supplied continuously to the vessel by a pump (16), while a piston (18) subjects the liquid medium to an alternating flow which ensures that the contents of each chamber (24) are well mixed and that the residence time for cells in the vessel (12) is substantially uniform. The residence time may be for example 24 or 48 hours. The cells can therefore be cultured continuously.

Description

Method for Performing Cell Culture

This invention relates to a method for performing cell culture, particularly but not exclusively for performing cell culture continuously.

It is known to perform fermentation and other cell culture processes in a vessel containing the cells and a suitable liquid medium, and the liquid may be stirred during the fermentation process with an impeller. Such a process is typically performed in a batch mode, and it is difficult to adapt such a vessel so the process can be performed continuously, because the residence time in such a mixed vessel is not uniform. A mixing apparatus for fluid media in which the residence time is more uniform is described in EP 0 229 139 B (N.R.D.C), comprising a long (e.g. cylindrical) vessel, means to set a fluid in oscillation along the vessel, and a series of shar -edged stationary obstacles (e.g. rings) along the vessel; the oscillation of the fluid generates vortices adjacent to the obstacles. Another mixing apparatus is described in GB 2 242 376 B (UKAEA) , in which reagents which form a precipitate, after being mixed using a fluidic vortex mixer, are passed through a vessel which comprises a plurality of substantially circular radiused sections connected back-to-back through which the mixed fluids are caused to oscillate so as to generate vortices .

According to the present invention there is provided a method of performing cell culture in which a suspension of cells in a liquid medium is enclosed in a vessel, the vessel comprising a plurality of equally spaced chambers forming a linear array, each chamber communicating at each end with the adjacent chambers in the array via respective linking ports of smaller cross -sectional area than the chambers, each port being defined by a smoothly rounded wall portion, and the liquid medium is subjected to an alternating flow along the vessel such that a vortex is formed in each chamber, and the direction of each such vortex alternates.

The alternating flow preferably varies sinusoidally with time. It causes the liquid in each chamber to flow in a vortex, the vortex expanding to occupy the chamber and then contracting again as a result of the velocity along the vessel increasing and then decreasing, and each vortex reverses direction as the flow along the vessel changes its direction.

The chambers and ports may be of circular cross - section, so the vortices are toroidal. Each chamber may be generally spherical, or may be cylindrical with a length (excluding the length of the adjacent port) preferably between about 0.5 and 1.0 times its diameter. The increase in diameter in passing through a port into a chamber must be abrupt enough to ensure flow separation; the radius of curvature ( in a longitudinal sectional plane) where the port opens out into the chamber is therefore preferably in the range a third to a hundredth of the radius of the chamber, for example about a quarter or a tenth the radius of the chamber. The linking ports are preferably of diameter in the range 0.4 to 0.8, for example 0.5, times the diameter of the adjacent chamber. The alternating flow preferably involves displacement of a volume of liquid at least 0.1, for example 0.8, times the volume of a chamber to ensure vigorous mixing .

Preferably the method also comprises superimposing a linear flow on the alternating flow of liquid through the vessel. The residence time for the liquid in the vessel is substantially uniform, varying by no more than about 20 percent, preferably varying by no more than 10 percent. Consequently the cells emerging from the vessel are at substantially the same stage in their growth. The linear flow and the alternating flow can be controlled independently of each other, so that a desired residence time can be achieved by selecting an appropriate linear flow rate. The mean residence time may for example be in excess of 6 hours, for example 48 hours or longer.

Preferably the method also comprises controlling the temperatures of the chambers in the vessel to optimize the cell growth, or to induce the production of a desired cell metabolite controlled by, for example, a thermally- induced genetic switch. The method may also comprise injecting liquids (such as cell nutrients) into one or more of the chambers in the vessel. The swirling of the vortices within the chambers ensures that all the contents are thoroughly mixed, but the cells are not subjected to high shear.

The vessel itself has no moving parts. It may for example be made of stainless - steel , and may be sterilised, for example with steam, before introducing the cell suspension. The vessel enables a cell growth process to be performed substantially continuously, by continuously supplying cells as an inoculum and appropriate liquid to the inlet end of the vessel and withdrawing cell suspension from the outlet end of the vessel.

The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawing which shows a diagrammatic view of an apparatus for cell culture. Referring now to the drawing, an apparatus 10 for culturing cells on a continuous basis comprises a vessel 12 (shown in longitudinal section) in which the cells grow for a substantially uniform time of 48 hours. A suspension of cells in a liquid medium containing appropriate nutrients is supplied to the vessel 12 via an inlet duct 14 by a mono pump 16 (indicated diagrammatically) , the mean flow rate rate through the pump 16 along with the volume of the vessel 12 together determining the residence time. The liquid in the inlet duct 14, and so too that in the vessel 12, is set in oscillation by an oscillating piston 18 driven approximately sinusoidally by a mechanical linkage 20 (indicated diagrammatically) . The vessel 12 has an outlet duct 22 at the opposite end from the inlet duct

14. The cells to be cultured are supplied as an inoculum into the liquid medium via an inlet duct 23.

The vessel 12 is of stainless -steel sheet defining a linear array of generally cylindrical chambers 24 on a common longitudinal axis, each chamber 24 communicating with the adjacent chamber or chambers 24 in the array via a port 26 which has a smoothly rounded profile in longitudinal section. Each chamber 24 has a length (excluding the ports 26) about 0.8 times its diameter.

The diameter of each port 26 is half the diameter of each chamber 24. As indicated by arrows A, the liquid in each chamber 24 flows in a toroidal vortex as a result of the liquid flow driven by the piston 18, because the increase in diameter in passing through a port 26 into a chamber 24 is sufficiently abrupt that flow separation occurs; in this case the radius of curvature (in a longitudinal plane) where the port 26 opens out into the chamber 24 is just less than a quarter of the radius of the chamber 24. The resulting vortex expands to occupy the chamber 24 as the liquid flow increases. When the oscillating piston 18 reverses direction this generates vortices in the chambers 24 with the opposite direction of swirl. These swirling vortices ensure that the liquid within any one chamber 24 is very thoroughly mixed. Vortex formation requires that at each port 26 the Reynolds number (at the maximum linear flow) is preferably above 100, more preferably above 500, and may be above 1000.

The vessel 12 is in a vertical orientation, and defines thirteen chambers 24 (only six of which are shown). It comprises a base module 28, three identical ^■ intermediate modules 29, and a top module 30, which are linked by flanges. Each module is enclosed by a heat transfer jacket 32 (only one of which is shown), so that the temperature of each module 28, 29 and 30 can be controlled. In addition inlet ducts with pumps 34 may be provided for each chamber 24 through which nutrients may be supplied (only one such inlet pump 34 is shown) . In one example each chamber 24 is of diameter about 0.3 m (and so of volume about 15 litres), and the volume of liquid displaced by the piston 18 is five litres. In another example each chamber 24 is of diameter about 0.1 m (and so of volume about 0.5 litres), and the volume of liquid displaced by the piston 18 can be varied between 0.1 and 0.2 litres.

The vessel 12 comprises thirteen chambers 24, but might comprise a different number of chambers 24. The apparatus 10 might indeed incorporate two or more such vessels 12 in series, the outlet from one vessel 12 being supplied to the inlet of the next vessel 12, and a single piston 18 driving the liquid in all the vessels 12 into oscillation. This enables longer residence times to be achieved than are practical with a single such vessel 12. The vessel 12 is shown in a vertical orientation, but it might instead be operated in a different orientation, for example horizontal .

In use of the apparatus 10 the vessel 12 is first cleaned and sterilised, and the lowermost chamber 24 is then filled with a liquid medium. The cell suspension is then supplied at a steady rate by the pump 16, and the liquid in the vessel 12 is set into oscillation by the piston 18. The vessel 12 is maintained at the appropriate temperature for optimum cell growth. After the first 48 hours the vessel 12 will be completely full-, and the liquid medium initially used to fill the lowermost chamber 24 will have left the outlet 22 of the apparatus 10, and subsequently the liquid emerging from the outlet 22 is the cell suspension which has undergone culture for 48 hours. As indicated by the line 35, the inoculum supplied to the inlet duct 23 may be tapped off from the outlet 22.

The apparatus may be used with a wide range of microorganisms. For example it may be used to brew beer. Alternatively it may be used for mammalian or insect cell cultures. It will be appreciated that the frequency and amplitude of liquid oscillation must be sufficient to ensure vigorous mixing, and that it may need to be different for different microorganisms, for example to ensure an adequate concentration of oxygen and nutrients in the liquid medium immediately adjacent to the cells (as cells growing at different rates will deplete their immediate environment of oxygen and nutrients at different rates). It will also be appreciated that the apparatus may be modified in various ways while remaining within the scope of the invention. For example the linear oscillation might be brought about by an air- driven pulse unit rather than a mechanically driven piston. The chambers 24 might be of a different shape, for example they might be generally spherical.

Not only may the apparatus be used to culture ) microorganisms in order to form desired compounds, but it may also be used to culture microorganisms in order to break down undesired chemical compounds. Thus it may be used to perform effluent treatment, in particular anaerobic treatment of liquid effluents.

Claims

1. A method of performing cell culture in which a suspension of cells in a liquid medium is enclosed in a vessel (12), the vessel (12) comprising a plurality of equally- spaced chambers (24) forming a linear array, each chamber (24) communicating at each end with the adjacent chambers (24) in the array via respective linking ports (26) of smaller cross -sectional area than the chambers (24), each port (26) being defined by a smoothly rounded wall portion, and the liquid medium is subjected to an alternating flow (18, 20) along the vessel (12) such that a vortex (A) forms in each chamber (24) and the direction of each such vortex (A) alternates.

2. A method as claimed in claim 1 which comprises superimposing a linear flow (16) on the alternating flow (18, 20) of liquid through the vessel (12).

3. A method as claimed in claim 2 wherein the mean residence time for the liquid in the vessel (12) is in excess of 6 hours.

4. A method as claimed in any one of the preceding claims which also comprises controlling (32) the temperatures of the chambers (24) in the vessel (12).

5. A method as claimed in any one of the preceding claims also comprising injecting liquids (34) into one or more of the chambers (24) in the vessel (12).

6. A method as claimed in any one of the preceding claims also comprising sterilising the vessel (12) before introducing the cell suspension.