This invention relates to the field of cell culture, and, in particular, to an apparatus for cell culture.

In cell culture, desired strains of biological cells are kept in appropriate physical, chemical and biological conditions for the purpose of their growth and multiplication. The main role of a cell culture apparatus is to maintain the environment for cell growth, which may necessitate the supply of various nutrients including gases, regulation of the temperature and prevention of contamination of the culture by undesired cells.

Cell culture has several applications, including the production of live cells, production of chemical substances including biomolecules for pharmaceutical use, and the chemical, biological or physical transformation of a substrate or medium on which they are grown.

An apparatus for the culture of biological cells in an aqueous medium must usually facilitate transfer of gaseous oxygen or carbon dioxide into the medium, whilst also facilitating removal of waste carbon dioxide or oxygen from the medium and mixing of the medium.

Mixing of the liquid medium may be required for several reasons. These include: to distribute dissolved gases and other nutrients and waste products, to keep the cells in suspension and to maintain a steady temperature throughout the medium.

Many types of biological cells, including mammalian cells, are delicate and are easily damaged by methods of mixing and aeration such as stirring and sparging. A stirring impeller, for example, can easily kill the cells.

Cell death due to bubbles largely depends on the size of the bubble. Small bubbles are much more damaging to cells than large bubbles. The reasons for this effect are thought to be that the collapse of a small bubble leads to large stress on a neighbouring cell, and that small bubbles are more effective than large bubbles at carrying cells to the surface and into a foam at the top of the medium. Foaming, caused by sparging with small bubbles, represents a problem because it interferes with the removal of gas via a sterile port at the top of the vessel, and kills cells as mentioned above.

To avoid the problems caused by sparging and stirring, several types of bioreactor have been invented (eg US6190913; WO2005049784). Some are based on shallow vessels in which waves are induced to encourage gas transfer and mix the medium. These shallow vessels are normally based on disposable plastic bags, while the waves are normally induced by rocking the platform supporting either the entire bag or a portion of the bag near the sides.

These "wave bag bioreactors" are effective at smaller vessel sizes (having a capacity of up to around 20 L) but have problems when scaled to larger vessel sizes. Firstly, the agitation becomes more difficult mechanically, because of the need for a larger rocking platform to move a larger volume of aqueous medium. Secondly, gas transfer and mixing does not scale favourably with the depth of the bag, so a large horizontal area is taken up by the bioreactor apparatus.

US2007037279 discloses a disposable bag bioreactor where the culture vessel is a "column bioreactor", having a substantially vertical tubular geometry. Large bubbles are injected intermittently at the bottom and rise stably because of the influence of the vessel walls. This type of bubble is known as a Taylor bubble or slug bubble. The width of the Taylor bubble is constrained by the walls of the tubular vessel. If the volume of air in the bubble is large enough, the bubble occupies almost the entire cross-section of the tube as it rises, with the liquid medium falling in a thin film on the vessel walls around the bubble. The bubble does not break up in flow, despite being arbitrarily large in volume.

In the apparatus of US2007037279, the width of "the single large bubble" is "50 - 99% of the tank width, preferably from 60 to 99%, more preferably 98.5%." In other words, this bubble is large enough to occupy the entire cross- sectional area of the column and is stabilised by the presence of the vertical tube walls. These Taylor bubbles have a relatively small surface area compared to small bubbles at the same volumetric gas flow rate. However, Taylor bubbles are very effective at mixing the liquid medium, since they are trailed by vortices that extend over the entire column width. A disadvantage of such cell culture systems is that, although the Taylor bubbles are stable against fission, the flow in their wake is relatively violent, and may cause cell death or foaming. Another disadvantage is that a relatively large flow rate of sterile gas may be required to aerate the culture.

M.R. Tredici et al. (in BioHydrogen, ed. Zaborsky et al. p391 , Plenum Press, New York, 1998) disclose a near-horizontal tubular bioreactor. However, no apparatus is provided for controlled injection of individual bubbles. Therefore this bioreactor exposes the culture medium to small bubbles and may form a foam in the vessel's headspace. In addition, the gas transfer and mixing conditions are not well controlled compared to our invention.

The high costs and difficulties associated with up-scaling cell culture, particularly of some types of cells such as mammalian cells, mean that there is a need for technologies that reduce these costs and scale-up difficulties. In the case of the culture of delicate cells with gaseous nutrient needs, such as mammalian cells, these difficulties are principally in achieving gas transfer and mixing without damaging the cells.

According to the present invention there is provided an apparatus for culturing biological cells, comprising:

a substantially tubular vessel containing, in use, a liquid cell culture medium;

at least one means for injecting, in use, at least one gas bubble into the vessel, the means for injecting being arranged such that the at least one injected gas bubble has a volume at least equal to the cube of a quarter of the diameter of the substantially tubular vessel; and

at least one means for removing gas from the vessel;

wherein, in use, the substantially tubular vessel is shaped and inclined relative to a horizontal reference plane such that the at least one gas bubble rises at a reduced speed relative to a bubble of the same volume rising in a bulk medium, and the bubble's volume remains approximately constant during the rise of the at least one gas bubble. In operation, the tubular vessel according to the present invention is filled with a liquid cell culture medium, and large bubbles of gas are injected at the bottom of the vessel and allowed to rise through the vessel. The gas collects at the top of the vessel where it is removed from an outlet port. A large bubble is one whose volume is greater than the cube of a significant fraction of the tube diameter.

The tubular vessel for cell culture according to the present invention has two main advantages over the prior art:

Firstly the presence of the tube walls stabilise large bubbles, so that the bubbles neither break up into smaller bubbles nor seed smaller bubbles from their edges. This contrasts with the behaviour of bubbles in a bulk fluid, where large bubbles are unstable and tend to split into multiple bubbles or seed small bubbles. For air bubbles rising freely in water at standard conditions, these effects occur for bubble diameters greater than approximately 0.2 m and 15 mm respectively.

Secondly, the gently sloping orientation of the tube ensures that the rise of the bubble along the tube is as gentle as possible. Unlike a large stable bubble in a substantially vertical tube, which can occupy almost the entire the tube cross section as it rises, a large bubble in a gently sloping tube occupies only around 50% of the tube cross section. Furthermore, the bubble rises much slower in the gently sloping tube than the substantially vertical tube, thus increasing gas transfer. Both the reduced cross section of the bubble and reduced velocity of the bubble reduce the shear rate on the culture medium moving past the bubble.

The effect of these two properties is that large bubbles in the vessel agitate and transfer gas into the medium, without causing significant enough shear stress or formation of small bubbles to damage cells.

The advantages of the controlled injection of large gas bubbles into the tubular vessel are to control gas transfer and mixing, while minimising the exposure of the culture medium to small bubbles, and to minimise foaming in the vessel's headspace. An example of the apparatus according to the present invention will now be described with reference to the accompanying drawings, in which:

Figure 1 schematically represents an apparatus for culturing biological cells according to the present invention;

Figure 2 shows another apparatus for culturing biological cells according to the present invention; and

Figure 3 shows another apparatus for culturing biological cells according to the present invention.

A first embodiment of the invention is shown in Figure 1. The culture vessel 1 is a closed tube made from a flexible sheet of a plastic such as polyethylene, containing a liquid cell culture medium 2. The vessel 1 is substantially tubular, i.e. in the vicinity of most elements of volume in the culture medium, the shape of the vessel 1 resembles that of a tube.

A tube is defined as the three dimensional shape that results when a closed curve on a two dimensional plane is swept through a contour line perpendicular to the closed curve, such that the following two conditions are satisfied: the closed curve has an aspect ratio close to unity, so that an approximate diameter may be defined as either of its dimensions; and the length of the contour line is much greater than the diameter of the closed curve.

The vector tangent at any point along the contour line defines the local axis of the tube. The tubular vessel 1 is constructed and oriented such that the angle of inclination between its local axis and the horizontal plane is low at most loci. This property of the tubular vessel 1 may be described as 'gently sloping'.

At the bottom of the vessel 1 is a gas inlet tube 3. A means of gas injection 15, such as a gas pump or valve connected to a gas pressure source is provided such that gas is made to flow through the air inlet tube 3 at a defined rate for a defined period of time, which creates a large bubble of a defined size 5. A defined time interval between bubbles ensures that they do not collide with each other. Each bubble, as it rises through the vessel 1 , is trailed by vortices 4 which mix the liquid across the entire tube width. The bubbles collect in the headspace 6 where they collapse. A gas outlet 7 vents the gas out of the headspace. The vessel is oriented at an angle of inclination a, which is between 0 and 45° in order to achieve the gentle agitation and reduced bubble cross section described above.

A second embodiment of the invention is shown in Figure 2, including the culture vessel 8, gas inlet 9, rising bubbles 10 and gas outlet 11. The culture vessel 8 is made from a tube that has been coiled into a helix. This shape allows a tubular vessel of a given volume to occupy a more compact space in three dimensions, while keeping a constant angle of inclination a. The minimum angle of inclination for this helical culture vessel is given by: tana = - (Eq. 1 )

2(D/d - \) where d is the diameter of the tube and D is the outer diameter of the helical culture vessel 8 as shown. If, for example, D = 1 m and d = 0.2 m, then a = 7.1°. If five helical turns of this tube are made to form a vessel approximately 1 m tall, the total capacity is approximately 400 L

A third embodiment of the invention is shown in Figure 3, including the culture vessel 12, gas inlet 13 and gas outlet 14. The culture vessel 12 is made from a tube that has been bent into a spiralling "staircase" shape. The staircase shape comprises straight sections that are joined by hairpin turns, such that the straight sections are stacked vertically into two rows, each row neighbouring the other in the horizontal direction. This shape also allows a tubular vessel of a given volume to occupy a more compact space in three dimensions while keeping an approximately constant angle of inclination.