GB2062481A - Stirring apparatus - Google Patents

Abstract

The apparatus comprises a stirring flask (5) with a stirrer (4) extending within the flask. The flask is held to the stirrer by means of a flexible coupling (6) which enables the other end of the flask to be moved in an orbital path by rotation of a disc (9) carrying an eccentric pin (19) engaging a dimple (21) in the flask base. The pin (19) is radially adjustable. The apparatus is particularly (but not exclusively) for stirring microcarriers in a liquid culture medium, wherein the microcarriers provide attachment surfaces to permit cell growth when the cells are of the "anchorage dependent" type. During stirring the stirrer remains stationary with respect to the moving flask and the resulting stirring action imparts vertical and horizontal components of force to the microcarriers. This enables suspension of the microcarriers to be achieved at lower stirring speeds than possible using conventional stirring techniques, thus improving cell yield. <IMAGE>

Description

SPECIFICATION Stirring apparatus The present invention relates to stirring apparatus and particularly to such apparatus which may be employed for use in stirring microcarriers or beads within a liquid culture suspension to enable the growth of anchorage-dependent living cells thereon.

The growth of living cells in a stirred liquid culture medium is well established and is important both in the research and industrial fields.

Mammalian cell growth such as in the case of cancer cells, can be generated in a stirred suspension culture without the need for the presence of an attachment surface. However, certain mammalian cell types exist which can only grow in the suspension being attached to a solid surface, and such cell types are termed 'anchorage dependent'.

In recent years, methods for growing anchorage dependent cells in suspension cultures have been developed using microcarriers, see for example U.S.

Patent Specification Serial No. 4036693. The use of such microcarriers is more economic relative to other known methods and provides environmental continuity of growth since the cells can be grown in a controlled environment which can be regulated to provide constant optimal cell growth.

Normal methods for cell growth in a liquid culture medium containing microcarriers involves the necessity to maintain the microcarriers in uniform suspension while avoiding damage to the growing anchorage dependent cells.

An accepted method of stirring microcarriers employs a horizontal revolving permanent magnet encased in.PTFE, suspended by some form of bearing about 10 mm above the bottom of a stirring.

Another known method, disclosed in U.K. Specification 1 485 741, employs a stirrer basically comprised of a stirring rod for immersion in the liquid to be stirred, the stirring rod being provided with means for imparting to it an oscillatory pivotal motion such that the end of the rod describes an orbital path within the liquid being stirred.

With such stirrers the only forces acting on the micro-carriers are horizontal centrifugal forces, with no vertical component until stirring speeds are exceeded such that turbulence occurs. At this stage, suspension of the microcarriers is achieved. When the microcarriers collide with each other however, during such turbulence, damage to the surface cell growth can be sustained, resulting in a lower yield than would otherwise be possible.

It is an object of the invention to provide stirring apparatus, particularly for stirring microcarriers in a liquid culture medium, having an improved stirring action which imparts both horizontal and vertical components of force to the liquid being stirred at low stirring speeds.

According to the invention there is provided stirring apparatus comprising a stirring vessel, a stirrer extending within the vessel, the vessel being held at one end to the stirrer by means of a coupling adapted to permit the other end of the vessel to be moved in an orbital path while maintaining the Stir- rer stationary with respect to the moving vessel.

The stirring action achieved with such apparatus imparts horizontal and vertical components of force to the stirred medium at low stirring speeds.

Thus when the apparatus is being used to stir microcarriers in a liquid culture medium, it is not necessary to stir until severe turbulence occurs to achieve suspension of the microcarriers. Suspension is attained in a more gentle manner due to the varying forces acting on the microcarriers at relatively low stirring speeds thereby avoiding the damage to cell growth experienced with prior art techniques at high, turbulent producing, stirring speeds.

Additionally since the vessel orbits and does not rotate around its axis during stirring, samples may be taken from the stirring vessel and injections may be made into the stirring vessel without the need to employ rotatable joints.

An embodiment of the invention will now be described by way of example with reference to the accompanying drawings wherein: Figure lisa side view of stirring apparatus according to the present invention; Figure 2 is a cross-sectional view of the lower portion of an embodiment of a stirring flask for use with the apparatus of Figure 1; and Figure 3 is a detailed cross-sectional view of the coupling for holding the flask to the stirring rod in the apparatus of Figure 1.

The stirring apparatus shown in Figure 1 comprises an inverted L-shaped support 1, rigidly mounted on a housing 2.

A rod 4 is rigidly attached, by means of a clamp 3, to the top of the support 1 and the rod 4 extends within a stirring flask 5 held to the rod 4 by a flexible coupling 6. The constructional details of the coupling 6 are shown in Figure 3 and will be described later.

The flask 5 is generally cylindrical having a neck 5" through which the rod 4 extends within the flask, and a rounded base 7. Two other necks 8 are shown positioned on a shoulder of the flask formed between neck 5" and the main cylindrical portion of the flask, and these may be used for injections into the flask or withdrawing samples as required. The flask 5 may be hermetically sealed and fully autoclavable.

A crank disc 9 has a shaft 10 rotatably mounted in bearings 11 positioned within the housing 2. The disc 9 is disposed above the housing 2 and beneath the flask 5, and the shaft 10 extends within the housing 2 through an opening 12 in its top surface 13, delimited by an annular wall 14. The axis of shaft 8 lies on the axis 4' of the stirrer rod 4.

The disc 9 has a downwardly depending skirt 9' which surrounds the wall 14 such that any spillage from the flask 5 is prevented from fouling the bear ings 11.

The end of shaft 10 extending within housing 2, is provided with a pulley 15. A motor 16 is installed within housing 2 and drives a pulley 17. The pulleys 15 and 17 are connected by belt drive 18 so that operation of motor 16 causes the crank disc 9 to rotate.

Means may be provided for actuating the motor and varying its speed. Such means will be easily recognisable to one skilled in the art and have thus not been illustrated or described in this disclosure.

The disc 9 is provided with a crank pin 19. The pin is positioned on a radius of the disc 9 and has a conical tip 20 which is engaged in a conical dimple 21 formed in the base 7 of flaskS.

The axis of the conical dimple lies on the axis 5' of the flask 5 which in the position shown, is inclined to the axis 4' of the rod 4. Thus during rotation of the disc 9 the base of the flask 5 is caused to move in an orbital path on the flexible coupling 6, and with respect to the rod 4 which remains stationary, such that the path traced by the axis 5' of the flask 5 is the generatrix of a cone.

The diameter of the orbital path of the base of the flask 5 may be adjusted by moving the position of crank pin 19. To this end the crank pin 19 may be adjustably positioned on the crank disc 9 such as by being slidable in a radial slot on the disc 9 and having means for anchoring the pin 19 at any desired position in the slot.

To achieve optimum suspension when stirring microcarriers in a liquid culture medium in the flask 5 of the apparatus described above, the diameter of the orbital path of the base of the flask is optimised to allow the rod 4 to be immersed to its maximum extent in the medium without touching the flask, and adjusting the speed of the motor 16. The forces thus acting upon the microcarriers in suspension have vertical and horizontal components due to the viscous drag imparted by the inner surface of the flask and the reaction of the stationary rod 4, causing a stirring motion. Thus it will be seen that during stirring with the arrangement described, the rod 4 attempts to prevent the contents of the flask from rotating while the movement of the flask attempts to achieve the opposite effect.

This results in the microcarriers continually changing their position relative to the flask. Each particle is therefore subjected to a varying acceleration in the horizontal plane due to the change of radius as the flask 5 orbits, and a varying vertical acceleration as points on the flask move up and down during orbit.

This results in an improved suspension at relatively low speeds of orbit.

Figure 2 shows an optimum shape of the lower portion of flask 5 having dimensions which enable total suspension of the particles at lowest rotational speed. The cylindrical portion of the flask 5 has a diameter D and the rounded base 7 is semi-circular in cross-section having a radius of curvature which is half the diameter of the flask 5. The conical dimple 21 is formed on the axis 5' of the flask as mentioned earlier, and the internal height of the dimple 21 is one twelfth the diameter of the flask 5, and the angle of the cone a few degrees greater than that of the cone-shaped tip 20 of the crank pin 19.

Other shapes of flask may of course be used having the cone-shaped dimple in their bases, or alternatively a flask holder may be employed for stirring flasks which do not incorporate a conical dimple acting as a bearing for the crank pin 19.

The flexible coupling 6 is shown in detail in Figure 3. The flexible coupling 6 comprises a silicone rubber-moulded diaphragm 22.

A threaded collar 23 having an annular flange 24 is dimensioned for close fit insertion in an aperture 25 of the neck 5" of the flask 5. The aperture 25 is surrounded buy a rim 26.

The diaphragm 22 is captivated between the flange 24 of collar 23 and a moulding 27, and this assemblage of parts is pulled hard against the upper surface of rim 26 by means of a nut 28 screwed up on the threaded collar 22 against the lower surface of rim 26.

The rod 4 is a tight fit through diaphragm 5 and may be of stainless steel, nylon or any other suitable material.

The arrangement of the flexible coupling 6 means that the coupling acts as a "universal joint" flexing to allow the flask to move in the above-described orbital path while the rod 4 remains stationary within the flaskS.

Although only one flask 5 has been shown positioned on the rotatable crank disc 7, nevertheless it will be appreciated that a number of vessels could be so mounted on the same disc, and operated by one machine by a multiplicity of crank assemblies such as crank pin 2 and cooperating dimple 21.

This arrangement could be driven by a single motor using either a multiple belt system or single belt interlacing each drive-wheel.

Although operation of the stirring apparatus has been described with reference to suspension of microcarriers in a liquid culture medium, nevertheless the invention is not limited thereto but has general application to mixing chemical substances, paint and other similar such operations.

Claims (17)

1. Stirring apparatus comprising a stirring vessel, a stirrer extending within the vessel, the vessel being held at one end to the stirrer by means of a coupling between the vessel and stirrer adapted to permit the other end of the vessel to be moved in an orbital path while maintaining the stirrer stationary with respect to the moving vessel.

2. Stirring apparatus as claimed in Claim 1 including rotatable means adapted to engage the vessel such that operation of said means causes the other end of the vessel to move in said orbital path.

3. Stirring apparatus as claimed in Claim 2 wherein said rotatable means comprises a rotatable disc to which is attached a crank pin engaging the vessel and lying on a radius of the disc.

4. Stirring apparatus as claimed in any preceding claim wherein said coupling comprises a flexible diaphragm extending across an opening in the vessel said stirrer passing through the diaphragm and in tight fit engagement therewith.

5. Stirring apparatus as claimed in Claim 4 wherein said opening is the entrance to a neck of the vessel formed at one end of a main cylindrical portion of the vessel, the other end of the cylindrical portion being semi-circular in cross-section having a radius one half the diameter of the cylindrical portion.

6. Stirring apparatus as claimed in any one of Claims 3 to 5 wherein the other end of the vessel is provided with a conical dimple, and said crank pin has a conical tip engaging in the dimple.

7. Stirring apparatus as claimed in Claim 6 wherein the axes of the conical dimple and the neck of the vessel lie on the axis of the main cylindrical portion of the vessel.

8. Stirring apparatus as claimed in Claim 7 wherein the height of the conical dimple is one twelfth the diameter of the main cylindrical portion.

9. Stirring apparatus as claimed in Claim 8 including a housing having an opening through which extends a shaft of the rotatable disc into engagement with bearings therefor installed within the housing.

10. Stirring apparatus as claimed in Claim 9 wherein said disc is provided with an annular skirt which surrounds an annularwall delimiting the periphery of the hole.

11. Stirring apparatus as claimed in Claim 10 wherein the shaft of said disc carries a pulley coupled by belt drive to a motor mounted within the housing thus providing means for rotating the shaft.

12. Stirring apparatus as claimed in any one of Claims 9 to 11 having a support member rigidly mounted on the housing for suspending the stirrer above the housing.

13. Stirring apparatus as claimed in any one of Claims 2 to 12 wherein the stirrer is a rod the axis of which lies on the axis of rotation of said rotatable means.

14. Stirring apparatus as claimed in any preceding claim wherein said stirring vessel is provided with one or more necks adapted to enable probe connections to be made to the vessel.

15. A method of stirring microcarriers in a liquid culture medium in a stirring vessel comprising mounting a stirrer within the vessel such that the vessel is held to the stirrer at one end, and moving the other end of the vessel in an orbital path while maintaining the stirrer stationary with respect to the vessel to stirthe contents of the vessel.

16. Stirring apparatus substantially as hereinbefore described with reference to the accompanying drawings.

17. A method of stirring microcarriers in a liquid culture medium substantially as hereinbefore described with reference to and as illustrated in the accompanying drawings.