WO1992006764A1

WO1992006764A1 - Method and device for separating cells from a fluid culture medium

Info

Publication number: WO1992006764A1
Application number: PCT/BE1991/000072
Authority: WO
Inventors: Alain Miller; Dominique Dubois
Original assignee: Alain Miller; Dominique Dubois
Priority date: 1990-10-10
Filing date: 1991-10-09
Publication date: 1992-04-30
Also published as: BE1004599A5; AU8621791A; IL99653A0

Abstract

A method for separating cells from a fluid medium, wherein a filter having a tubular shell for filtering out at least a fraction of said cells is immersed in said fluid medium, said filter is rotated about its longitudinal axis, a filtrate inside the filter shell is separated by filtration from a residue outside said filter shell, the filtrate is drawn out of the filter, and, during filtration, the fluid medium is stirred in a portion thereof which is located at the periphery of the tubular filter shell. A device for implementing the method is also provided.

Description

"Method and device for separating cells from a fluid culture medium"

The present invention relates to a method for separating cells from a fluid culture medium, comprising an immersion in the fluid medium of a filter having a tubular envelope capable of filtering at least a fraction of said cells, a entrainment of this filter in a direction of rotation about its longitudinal axis, separation by filtration of a filtrate inside the filter casing and a retentate outside of it, a withdrawal of the filtrate out of the filter, and agitation of the fluid medium, simultaneously with filtration. It also relates to a device for separating cells from a fluid culture medium, comprising a tank in which the fluid medium is contained, a means for filtering the fluid medium in the form of a filter immersed in the latter and having a filtering tubular casing, capable of filtering at least a fraction of said cells, means for rotating this filter around its longitudinal axis, means for withdrawing from the filter a filtrate which has reached the inside of the tubular filtering casing, and a means for agitating the fluid medium, capable of operating simultaneously by means of filtration.

The invention relates in particular to the agitation of fluids used in fermentative processes in a bioreactor, and the retention of a biomass inside the bioreactor. It allows in particular to concentrate the biomass inside the bioreactor while maintaining a continuous and efficient mixing of the fluid.

By cells is meant the cells themselves (microorganisms, animal or plant cells, isolated or associated), or a matrix serving to support the cells (in particular macroscopic supports, full or hollow microsupports, carrageenan beads or d alginate, encapsulation matrices, particles serving as a substrate nutrient with which cells are associated).

By separation of cells is meant, starting from a fluid containing cells within the meaning of the invention suspended or suspended by stirring, obtaining two fluids which can be collected separately, one containing cells and the other does not or one which differs from the other by the size and / or the concentration of cells which it contains in suspension.

The invention relates more particularly to the stages of culture and concentration of animal cells in a bio-reactor, for the production of different types of biological materials. As examples of such materials obtained from cell culture, mention may be made of the cells themselves, the cellular components, the viruses or viral proteins, the viral or bacterial vaccines, the monoclonal antibodies, the proteins resulting from the manipulation of the cell genome. In view of the large and growing demand for such products in human or veterinary medicine and in molecular biology, the invention will be described below with reference to the culture of animal cells.

A bioreactor is an enclosure in which the cells are subjected to an environment which can be controlled, and which corresponds to the physiological requirements of the cells: the parameters controlled are for example the temperature, the supply of nutritive agents, the withdrawal of products from the metabolism, oxygenation of the culture medium, acid-base balance, oxidation-reduction balance, turbulence and shear forces, cell concentration, pressure.

Each of these parameters depends on the system for agitating the medium, as well as on the system for introducing culture medium into the tank, or for removing used medium from the tank. It follows that the agitation of the fluid and the renewal of the fluid are two major operations for the success of cell culture. Given the great sensitivity of cells to variations in their environment, it is desirable, in a process of producing biological materials, that the cells be kept as long as possible inside the bioreactor. There are known devices for cultivating animal cells in a stirred tank in which isolated cells or anchoring supports for the cells are suspended. According to the conventional methods used in these devices, the stirring is carried out by a stirring mobile immersed in the fluid, and the mobile is set in motion by a motor, or contains a magnetic bar set in motion by a magnetic stirrer ( small volume tanks) (A. PROKOP et cons., Bioreactor for ammalian Cell Culture, in FIECHTER A., Advances in Biochemical Engineering / Biotechnology, 39, p. 29-71, Springer-Verlag, Berlin, 1989).

All the parts in contact with the culture are sterilized before inoculation, and must remain sterile during the culture.

The expected performances of the stirring system are in particular the maintenance in suspension of the biomass, the homogenization of the concentrations in the fluid, the contact between phases

(gas-liquid-biomass), aeration of the culture medium.

However, such devices do not allow a separation between the biomass and the spent liquid, in the tank itself. These separation steps are however essential, both in the processes which aim at the production of cells and in the processes which aim at the production, by cells, of solubiated substances. In a method of cultivating a batch of cells in a bioreactor, after the cell culture phase, the liquid volumes to be treated are very large relative to the harvested weight of concentrated biomass. Typically, 10 liters of a culture medium containing 1.6 million animal cells per milliliter of medium must be centrifuged, in order to harvest only 50 grams of cells (weight of the wet pellet). These centrifugation (or microfiltration) steps made necessary for the separation of the particles, require investment, manpower and compliance with safety instructions.

In cases where the production of biomass is the aim of the process, these steps, performed outside the bio-reactor, alter the quality of the cells harvested, and are likely to increase the quality variation from batch to batch. 'other. Now, maintaining on the contrary, the quality of the cells harvested is particularly important in the mass production of animal cells intended for the structural study or for the study of the functional activity of cellular constituents. In the case of cell culture for the culture of pathogenic organisms (for example production of viral vaccines), the infectious risks run by the personnel are linked to the number of steps necessary downstream of the bioreactors. Furthermore, the concentration of cells inside the bioreactor is appreciated in the production of virus, at the stage of cell inoculation by the virus, because the viral inoculum is not diluted in a large volume. liquid medium.

In both cases, maintaining the sterility of the production line is made difficult by the volume of bioreactor effluents to be treated.

The substantial concentration of cells inside the bioreactor itself constitutes for these reasons an appreciable reduction in the investment and operating costs of the first steps downstream of the bioreactor, an improvement in the safety of particle separation, an improvement in the quality of the cells harvested, and a reduction in the total duration of the process.

We then developed, for the continuous culture of cells, tanks containing a filter rotating around an axis. This filter is usually used to isolate within the bioreactor a compartment essentially devoid of cells and into which one or more fixed tubes are introduced, to pump the liquid medium out of the bioreactor (see inter alia S. REUVENY and cons.,

Comparison of cell propagation methods, 3ournal of Immunological

Methods, 86, 1986, p. 61-69; P.5. THAYER, Spin Filter Device for Suspension Cultures, in KRUSE PF et cons., Tissue Culture Methods and Applications, Académie Press, 1973, p. 345-351). In these devices, however, the agitation of the culture medium results solely from the turbulence caused by the rotation of the filter, which is insufficient or inappropriate in many circumstances. We have also made devices in which the growth and the agitation of the culture medium take place in a main tank and the filtration of the culture medium in an auxiliary tank (see WR TOLBERT et cons., Perfusion culture Systems for production of mammalian cell biomolecules, in J. FEDER et al., Large-scale mammalian that culture, Académie Press, 1985, p. 97-119). Such an arrangement proves to be particularly expensive, and has the abovementioned drawbacks which are inherent in all the devices where the separation must take place downstream of the culture vessel. We have also developed a process and a device as described at the beginning (see, among other patents US-A-. 166,768; P. HIMMELFARB et al., Spin Filter Culture ..., Science, vol. 1, p. 555 -557; GC AVGERINOS et cons., Spin Filter Perfusion ..., Bio / Technology, vol. 8, 1990, p. 54-58). These devices are used in continuous culture in perfusion, to maintain the cells in the bioreactor and to increase the cell concentration during continuous culture. However, they are not used to concentrate the cells in the bioreactor at the end of a batch culture. In reality the rotary filters are placed above the stirring mobile, consists of a magnetic rod in small tanks or a pumping mobile in large tanks. However, it is not possible to lower the liquid level in the bioreactor below the lower level of the rotary filter, and the latter is relatively high in the tank given the presence of the underlying agitator. The presence of the filter, bringing the stirring wheel down, impairs its pumping efficiency. The agitation of the culture medium in the entire tank from an area located at the bottom thereof collides with the sensitivity of the cells to shearing forces.

The present invention aims to develop a method and a device for cell separation, which do not have the drawbacks of the methods and devices according to the prior art. She must . in particular to allow the separation of cells by remedying the agitation difficulties encountered with current means. Advantageously, this process must be able to be applied to the various modes of culture. existing, traditionally classified according to the evolution of contributions and withdrawals of medium to the bioreactor. In order to show the extent of the applications of the method and the device according to the invention, these various cultivation methods can be described below. 1. - In batch culture, the nutritive agents are provided by the culture medium present during inoculation, the culture then evolving without substantial addition or withdrawal of culture medium, until the end of the culture. of the lot. The volume of the culture is constant. 2. - In batch culture, there is a continuous or discontinuous supply of culture medium to the bioreactor, without substantial withdrawal of liquid from the bioreactor. The volume of culture is increasing. 3. - In continuous culture, there is an addition of culture medium in conjunction with a withdrawal of liquid from the culture. The volume of the culture is generally kept constant. When, in particular, a culture medium is removed from the bioreactor by associating with it a method of cell separation, which retains the cells in the bioreactor or returns the cells to the bioreactor, the continuous culture is said to be in perfusion. This results in an increase in cell concentration in the bioreactor.

To resolve these problems, a separation method as described at the outset is provided, this method comprising agitation of the fluid medium from a zone thereof which is situated at the periphery of the filtering tubular envelope. Advantageously, provision is made to immerse the filter to the bottom of the agitated fluid medium so as to obtain filtration over the entire height of the fluid medium. According to a particular mode of the method according to the invention, said zone, from which the agitation of the fluid medium takes place, is situated along the entire height of the submerged filtering tubular envelope. According to a preferred embodiment of the invention, the agitation produces a tangential circulation of the fluid medium over the height of the submerged filtering tubular casing. According to the invention, the stirring can produce a simultaneous transfer of at least one gas, present above the fluid medium, inside the latter and a distribution of this or these gases in the fluid medium. According to the invention, there is also provision for a separation device as described at the start, this device comprising a means for agitating the fluid medium, arranged around the tubular filtering envelope of the filter. According to an advantageous embodiment of the invention, the stirring means is a helical ribbon arranged around the tubular filter envelope, optionally over the entire height thereof. According to a preferred embodiment of the invention, the helical tape is fixed in places on the tubular filter envelope, with an internal edge of the tape located a short distance from said envelope, in particular of the order of 0.5 to 5 mm. According to the invention, the helical ribbon can in particular be arranged so as to be able to partially emerge above the fluid medium. According to another embodiment of the invention, the stirring means is formed of blades driven in rotation around the above-mentioned filtering tubular casing. Very advantageously, the filter is immersed up to a small distance from the bottom of the tank, in particular of the order of 1 cm.

Other details and particularities of the invention will emerge from the description given below, without implied limitation and with reference to the attached drawings.

Figure 1 shows, schematically, a sectional view of a device according to the invention.

2 shows a sectional view of a mechanism for driving the rotary filter and the stirring means, in an alternative embodiment of a device according to the invention.

Figure 3 shows, schematically, a sectional view of yet another embodiment of dispo¬ device according to the invention.

The figure shows a detailed view of a filter support in a device according to the invention.

FIG. 5 represents a view in section along the line V-V of FIG. 1.

In the different figures, identical elements have the same references. As shown in Figure 1, the device illustrated comprises a tank 1 in which is contained a fluid culture medium 2. This tank 1 is closed at its upper end by a closing plate 3. This supports a motor 4 which, in service, rotates an output shaft 5 in the direction of rotation indicated by the arrow FI. A rotary filter 6 is mounted on this shaft. It comprises an upper support flange 7 and a lower support flange 8 which are mounted on the shaft 5 so as to be driven in rotation by the latter. Between the two flanges 7 and 8 is stretched a filter envelope 9 in the form of a cylindrical mesh. The filter may consist of a stainless steel mesh of known nominal and absolute fineness. Alternatively, the mesh can be made of porous ceramic or a synthetic polymer of good biological compatibility and resistant to sterilization, in particular nylon, ethylene-tetrafluoroethylene (ETFE).

The trellis is closed on itself to adopt the shape of a cylinder with a vertical axis, and it is therefore integral with the rotation shaft 5 of the motor 4 placed at the top of the tank.

The particle separation performance of the lattice is expressed by the size of the cells retained in a functional test (nominal separation diameter), rather than by the diameter of the spaces formed between the lattices of the lattice. Indeed, the spaces provided by the trellis do not necessarily have a circular shape. The choice of the fineness of the mesh of the rotary filter is a compromise between the diameter of the cells to be retained and the operating conditions under which the separation will take place.

Indeed, the diameter of the cells retained by the rotating rotary filter depends on the nominal diameter of separation of the lattice, its tangential speed, the possible flow of fluid through the lattice, the configuration of the stirring system (shape of the lattice , geometrical relationships between the stirring means and the lattice), the appearance of soiling (tendency of biological media to form deposits on surfaces and to plug orifices of small diameter), and of the interactions existing between suspended cells and the lattice or the substances deposited therein (in particular hydrophobic, electrostatic interactions, electrocine¬ ticks).

Consequently, the rotary filter may possibly be equipped with a trellis having a nominal separation diameter greater than the size of the cells to be separated, since the effective cut-off diameter of the rotating trellis is greater than the cut-off diameter determined by a functional test on static trellis.

According to the invention, the stirring means is arranged around the filter envelope, that is to say at the same height as the filter envelope, which makes it possible to improve the agitation and filtration functions. , as described below. The size of the two elements can be such that they extend over the major part of the height of the phase containing the cells, and they can exert a stirring and filtration function over most of this phase.

In the embodiment illustrated in the figure

1, the stirring means consists of a helical ribbon 10, disposed around the filter envelope 9 in the form of a cylindrical trellis.

In this example, the helical ribbon is fixed in any suitable manner at one of its ends 12 to the upper support flange 7 of the filter and at the other end 13 to the lower support flange 8 of the filter.

By its rotation by means of a motor arranged above, or possibly also below the tank, this agitator ensures particularly efficient pumping of the fluid, with generation of a minimum turbulence field in the fluid. When it is a question of agitating cells sensitive to the shear forces, the various functions of agitation described above (maintenance in suspension, homogenization, contact between phases, aeration) are best encountered by a type of agitator which achieves maximum pumping for minimum turbulence. In tanks agitated in this way, the liquid streams 11 (Fig. 1) describe a generally vertical trajectory over the entire height of the tank; more particularly, the liquid is pumped downwards by the stirring means 10 and it rises along the walls of the tank, to be pumped back into the stirring means. Such circulation in the vertical axis of the tank defines an axial type agitator.

Among the axial type agitators, the helical ribbon and the Archimedes' screw exert greater pumping than a marine propeller or a turbine with inclined blades. In the case of operation in turbulent regime, this type of mobile also provides greater homogeneity of the turbulence field, than a radial type agitator. Typically, the helical ribbon 10, arranged vertically so as to surround the cylindrical trellis

9, is immersed to the bottom of the tank, and it ensures the pumping of the fluid downwards.

The material chosen for the production of the stirring means must meet the requirements of the process, and will in particular have good mechanical strength, good biological compatibility, resistance to sterilization conditions (thermal sterilization in autoclave or in situ). Mention may in particular be made of synthetic polymers, stainless steel, composite materials formed of epoxy resins reinforced with carbon, aramid or glass fibers.

When the transfer of gases from the surface to the liquid is an important parameter, it is preferable that the upper part 12 of the helical ribbon 10 be emerged from the fluid medium 2, so that pumping downwards increases the coefficient of transfer of gases. towards the liquid phase. This is particularly the case in the culture of animal cells in a bioreactor, where oxygen is supplied to the cells from the surface of the liquid, or from fine submerged tubes permeable to gases. In these two aeration modes, it is preferable that the stirring system ensures a high pumping rate, without however creating a turbulent regime. The supply of oxygen to the cells is a critical factor, given the very low solubility of oxygen in aqueous media, the sensitivity of the cells to variations in dissolved oxygen, and the sensitivity of the cells to the shearing effects created. by gas bubbles. of used fluid, and the concentration of cells in situ, can be exerted in partially filled tanks, or in tanks for which the height of the level of the liquid can vary during culture. A very specific embodiment example of a device according to the invention, of the type illustrated in FIG. 1, will now be described.

The filter casing 9 consists of a mesh of braided stainless steel wires, of the T illed Dutch eave type (Haver & Boec er, R.F. Germany). The mesh chosen has a nominal fineness of 10 micrometers, and an absolute fineness of 15 to 18 micrometers. Depending on the type of cells to be separated, the fineness of the filter to be chosen may be very different, for example between 0.5 micrometers and 1 centimeter, but in general it is between 5 micrometers and 200 micrometers.

In the case of animal cells in suspension, the average diameter of these is typically 13 micrometers. It is possible to choose a trellis having a nominal separation diameter greater than the size of the cells to be separated, because the effective cut-off diameter of the rotary filter is greater than the cut-off diameter determined on the trellis under static conditions.

The trellis is closed on itself to adopt the shape of a cylinder with vertical axis (variable diameter from 3 centi¬ meters to several meters, depending on the dimensions of the tank, typically from 4 to 8 cm for a tank of 15 liters with a height of 44 cm and an internal diameter of 22 cm), and it is fixed to a stainless steel support by laser welding.

A type of support is shown more particularly in FIG. 4, in which the trellis has not been drawn, for reasons of clarity of the drawing.

The trellis is carefully welded to its lower base on the flange 8 so as not to leave space between the support and the trellis. The flange 8 has a central rod 27 which, by a threaded axial bore, secures the threaded end of the rotation shaft 5, and which extends this shaft so as to The illustrated device further comprises a fixed suction tube 25 which opens into the interior of the filter 6, at a level close to the bottom of the tank, and which is therefore capable of withdrawing the filtrate which has reached the interior of the filter. The device also comprises a fixed suction tube 26 which opens at the bottom of the tank 1 and outside the filter 6, and which is capable of withdrawing from the retentate. A tube for introducing fresh fluid medium 28 can also be provided to supply the retentate retained in the tank 1 outside the filter 6. According to the embodiment illustrated in FIG. 2, the filter 6 partially shown and of schematically tick and the stirring means not shown are not integral with each other. The output shaft 5 carries a coupling mechanism 14 of the type having planets 17 and 18, which pivot around the longitudinal axis of the shaft 5, and satellites 15 and 16 which pivot around the axis of shaft ends 20, supported in any way for example by the closure plate of the tank. The sun gear 17 is rotated in the direction FI by the output shaft 5 and, via the satellites 15 and 16, it rotates in the opposite direction F2 the sun gear 18 and the shaft 19. The shaft 19 is, in the example illustrated, coaxial with shaft 5 and they each rotate in opposite directions. The filter 6 is supported by the sun gear 18 and rotates in the direction F2. By cons, the stirring means, for example a helical ribbon, can be fixed to the shaft 5 by a support flange not shown, of the type of the flange 8 in Figure 1. It then rotates in the direction FI.

It is obvious that one can also provide a reducing or multiplying coupling mechanism making it possible to drive the filter and the stirring means at different speeds. It is also possible, in all of the embodiments according to the invention, to provide for modifying the rotation speed of the filter as a function of predetermined conditions.

According to the embodiment illustrated in FIG. 3, the device according to the invention comprises a radial type agitator, constituted for example by one or more discs 21 carrying at their periphery straight blades 22. These discs are mounted so as to be driven in rotation by the output shaft 5 of the motor 4. In the illustrated embodiment, the disc 21 is supported on the lower support flange 8 by support rods 23 arranged vertically around the filter 6. It is obvious that other methods of fixing the discs can be envisaged, for example through the filter envelope 9.

Such a device is used in particular when it is desired to obtain greater turbulence in the fluid. In this type of device, the streams of liquid current 24 leave the agitator radially towards the wall of the tank 1, and they are divided into two parts: the upper one goes up along the wall, then is pumped into the agitator, the other, lower, descends along the wall towards the bottom of the tank, then is pumped into the agitator. When it is desired to operate the device in liquid level tanks of variable height, it is useful for the disc 21 located at the bottom to be close to the bottom of the tank.

The devices which have just been described can be used in particular, although not exclusively, in a culture of animal cells in an appropriate culture medium.

While maintaining the turbulence at a minimum value, the device according to the invention allows efficient agitation of the culture medium in the various culture modes described previously. The method makes it possible to concentrate the cells at the end of a batch culture, while maintaining them in the adequate and controlled physiological environment present in the bioreactor. The liquid volumes to be treated in order to harvest the cells are greatly reduced. This concentration process is made possible by the use of a separation device, the filter element of which can fall to the bottom of the tank.

By continuous or intermittent withdrawal of used medium essentially devoid of cells, the device according to the invention allows continuous culture at high cell concentration. The various functions of agitation, withdrawal of used fluid, and the concentration of cells in situ, can be exerted in partially filled tanks, or in tanks for which the height of the level of the liquid can vary during culture. A very specific embodiment example of a device according to the invention, of the type illustrated in FIG. 1, will now be described.

The filter envelope 9 consists of a mesh of braided stainless steel wires, of the Twilled Dutch eave type (Haver & Boecker, R.F. Germany). The mesh chosen has a nominal fineness of 10 micrometers, and an absolute fineness of 15 to 18 micrometers. Depending on the type of cells to be separated, the fineness of the filter to be chosen may be very different, for example between 0.5 micrometers and 1 centimeter, but in general it is between 5 micrometers and 200 micrometers.

The trellis is carefully welded to its lower base on the flange 8 so as not to leave space between the support and the trellis. The flange 8 has a central rod 27 which, by a threaded axial bore, secures the threaded end of the rotation shaft 5, and which extends this shaft so as to position the stirring means 10 at the bottom of the tank. On the flange 8 is welded a support tube 31 of stainless steel of the same diameter, very much openwork. The trellis follows the cylindrical shape of the perforated tube 31, and the upper edge of the trellis is welded to the upper end of the tube. Alternatively, the upper edge of the treilis can be welded to an upper flange, as illustrated in FIG. 1.

The rotary filter thus described is closed at its base, it has a trellis over its entire height, and it is open at its top. The flange 8 which forms the base of the filter can also be perforated and support a horizontal trellis. In this case, the filter comprises a plane trellis at its base and a cylindrical trellis over its entire height.

One or more fixed tubes 25 (for example 6 millimeters in outside diameter) are arranged vertically in the interior space of the filter, and their end is close to the bottom of the latter. These tubes make it possible to withdraw the fluid having passed through the filtering element, or to introduce various fluids, or else to apply specific conditions for treating the filtrate. H are attached to the closure plate 3 (Figure 1). Alternatively, tubes can be held integral with a horizontal stainless steel intermediate plate 32, which bears on parts not shown of the bioreactor, and the tubes are connected to the closure plate 3. The horizontal section of the trellis described here is a simple circle, but it can be modified in particular in a scalloped circle, in order to locally increase the turbulence.

In a tank of 15 liters of total volume (height 44 cm, internal diameter 22 cm), used at 9 liters, the filter element typically has the following dimensions: height of the filter cylinder: 250 mm diameter of the filter cylinder: 60 mm height of the submerged part of the filter cylinder: 220 mm submerged surface of the mesh: 415 cm ² elevation of the filter compared to the bottom of the tank:

I cm. This distance (designated by the reference D in Figure 1) allows for example the passage of a drain tube. of the tank. The stirring means consists of a helical ribbon 10 disposed around the cylindrical trellis, and traversing its entire height. The helical ribbon is composed of modified epoxy resin (Fibredux 914, Ciba-Geigy, Switzerland) pre-impregnated with unidirectional carbon fibers (High tensile Carbon T300). For its realization, we can consider for example that Fibredux ribbons

914 are cut and placed on a Duralumin mold cut in a lathe, and heated under pressure so as to form a reinforced composite material having the following characteristics:

- high mechanical strength (shear strength in laminar inland ILSS = 80-90 MN / m ^z at 37 ° C; resistance to rupture by bending, longitudinal (0 °) UFS = 1700 MN / m ² ; flexural modulus FM = 125 GN / m ² ; resistance to flexural rupture, transverse (90 °) TFS = 95 MN / m ² ; transverse flexural modulus TFM = 7.5 GN / m ² )

- resistance to thermal sterilization (121 ° C, 1.5 bar, steam, 30 minutes)

- biological compatibility with animal cells and having the following dimensions: height of the helical ribbon: 250 mm pitch of the helical ribbon: 140 mm width of the helical ribbon: 42 mm internal diameter: 61 mm, external diameter: 145 mm thickness of the helical ribbon: + _ 0.9 mm

The upper and / or lower edge of the helical ribbon is made integral with the rotary filter by one or two removable metal parts 29 and / or 30, fixed to the upper and / or lower part of the rotary filter.

It is useful to keep this agitation and filtration unit in constant rotation, so as to reduce the appearance of possible deposits and the contamination of the mesh. This is particularly the case in biological fluids which are strongly loaded with dissolved substances (proteins, lipids, DNA molecules) and particles capable of clogging the trellis.

The agitation and filtration unit prevents or delays the possible appearance of soiling in highly charged fluids, taking advantage of the following factors:

1. The arrangement of the stirring means around the trellis increases the local turbulence at the location of the trellis. On the contrary, the turbulence prevailing at the level of the usual rotary filters is low, because the stirring means is arranged below the rotary filter, and the circulation in the tank is essentially of the laminar type.

2. The distance of 0.5 to 5 mm, existing, according to the invention between the rotary filter and the helical ribbon and designated in Figure 5 by the reference d, allows a tangential flow of liquid over the height of the rotary filter. This arrangement increases the turbulence at the level of the filter, and prevents the appearance of zones of low circulation between the filter and the helical strip.

3. The configuration of the trellis extending from the bottom of the tank to the surface of the liquid, gives a large filtration surface. The usual rotary filters have on the contrary a reduced height compared to the dimensions of the tank.

The choice of a helical ribbon as a means of agi¬ tation provides maximum pumping: the agitation carried out can be quantified by the oxygen volume transfer coefficient (parameter kL.a). In a 15 liter tank filled with 8 liters of liquid and aerated by the surface, the value of kL.a obtained with the device according to the invention described, rotated at 100 revolutions per min., Is 4.0 / hour, against a value of + _ 0.5 / hour obtained with a conventional marine propeller provided for this type of tank.

The pumping efficiency can be further increased by adding a segment of immersed concentric fixed tube, wider than the stirring means and of lower height. This guide tube, known per se and not shown, allows the entry of the fluid by its upper edge and the exit of the fluid from the pumping zone, by its lower edge. Such a guide tube allows the use of the device according to the invention for agitating the medium in the batch and continuous culture. Such a guide tube can typically have a diameter of 18 cm and a height of 12 cm, for a tank whose height of the liquid level is 23 cm. By way of example, the device according to the invention which has just been described has been used for the concentration of cells cultured in batches, at the end of culture. It allows in one hour to reduce from 10 liters to 1 liter the volume of a culture to 3 million animal cells per milliliter, by withdrawing the filtered medium at a rate of 150 milliliters per minute (filtrate). Using a stirring and filtration unit in rotation at 150 revolutions per minute, a nominal cut-off diameter of 10 micrometers and cells having a size distribution mode of 13 micrometers, the concentration of cells in the filtrate is three orders of magnitude less than the concentration in the retentate. In the example cited, the effluent containing the concentrated cells (retentate) comprises one liter of cells at 29.7 million cells per milliliter.

It should be understood that the present invention is in no way limited to the forms and embodiments described above and that many modifications can be made thereto without departing from its scope.

One can for example provide for the implementation of the method according to the invention in a culture medium of cells hung on a support suspended, for example microbeads, and filtration of the cells detached from their support.

Claims

1. A method of separating cells from a fluid culture medium, comprising immersing in the fluid medium a filter having a tubular envelope capable of filtering at least a fraction of said cells, driving this filter in one direction of rotation about its longitudinal axis, a separation by filtration of a filtrate inside the filter casing and a retentate outside of it, a withdrawal of the filtrate from the filter, and a agitation of the fluid medium, simultaneously with filtration, characterized in that it comprises agitation of the fluid medium from a zone thereof which is located at the periphery of the filtering tubular envelope.

2. Method according to claim 1, characterized in that it comprises immersing the filter to the bottom of the agitated fluid medium so as to obtain filtration over the entire height of the fluid medium.

3. Method according to either of claims 1 and 2, characterized in that said zone, from which the agitation of the fluid medium is effected, is located along the entire height of the submerged filter tubular casing. .4. Process according to any one of claims 1 to 3, characterized in that the agitation produces a tangential circulation of the fluid medium over the height of the submerged filtering tubular envelope.

5. Method according to claim 3, characterized in that the agitation produces a simultaneous transfer of at least one gas, present above the fluid medium, inside the latter and a distribution of this or these gases in the fluid medium.

6. Method according to any one of claims 1 to 5, characterized in that, to produce agitation, it comprises a rotary drive of a stirring means arranged around the filter, in a direction of rotation different from that of the filter.

7. Method according to any one of claims 1 to 5, characterized in that, to produce agitation, it comprises a rotary drive of an agitation means arranged around the filter, in the same direction of rotation than that of the filter.

8. Process according to any one of claims 1 to 7, characterized in that it comprises a modification of the level of the fluid medium during the process. 9. Method according to any one of claims 1 to 8, characterized in that it comprises a supply of fresh fluid medium to the fluid medium during agitation and / or separation.

10. Process according to any one of claims 1 to 9, characterized in that it comprises a drive of the filter in rotation at a variable speed.

11. Process according to any one of claims 1 to 10, characterized in that it comprises a culture of a batch of cells and, at the end or during the culture of the batch, the filtration of the stirred medium, with concentration of cells in the culture medium itself.

12. Process according to any one of claims 1 to 10, characterized in that it comprises a continuous culture at a high cell concentration in the retentate.

13. A method according to any one of claims 1 to 12, characterized in that it comprises a culture of cells hung on a suspended support, and a filtration of the cells detached from their support.

14. Device for separating cells from a fluid culture medium (2), comprising a tank (1) in which the fluid medium is contained, a means of filtration (6) of the fluid medium in the form of a filter immersed in the latter and having a tubular filtering envelope (9), capable of filtering at least a fraction of said cells, means for driving (4, 5, 15-18) this filter in a direction of rotation (FI, F2) around its longitudinal axis, and means for withdrawing (25) from the filter a filtrate which has reached the interior of the filtering tubular casing (9), and a means of agitation (10, 21, 22) of the fluid medium, capable of operating simultaneously with the filtration means, characterized in that the agitation means (10, 21, 22 ) of the fluid medium (2) is arranged around the tubular filter envelope (9).

15. Device according to claim 14, caracté¬ ized in that the stirring means is a helical tape (10) disposed around the tubular filter envelope (9), optionally over the entire height thereof.

16. Device according to claim 15, caracté¬ ized in that the helical tape (10) is fixed in places on the filtering tubular envelope (9), with an internal edge of the tape located at a short distance (d) of said envelope, in particular of the order of 0.5 to 5 mm.

17. Device according to one of claims 15 and 16, characterized in that the helical strip (10) is arranged so as to be able to partially emerge above the fluid medium (2). IS. Device according to claim 14, caracté¬ ized in that the stirring means is formed of blades (21) driven in rotation around the above-mentioned filtering tubular casing (9).

19. Device according to any one of the claims 14 to 18, characterized in that the filter is immersed up to a small distance (D) from the bottom of the tank (1), in particular of the order of 1 cm. .

20. Device according to any one of the claims 14 to 19, characterized in that it comprises means for driving in rotation (4, 5, 15-20) the stirring means in a different direction (FI) the direction of rotation (F 2) of the filter. 21. Device according to any one of the claims 14 to 19, characterized in that it comprises means for driving in rotation (4, 5) the stirring means in the same direction as the direction of rotation (FI ) of the filter.

22. Device according to any one of claims 14 to 21, characterized in that it comprises means for driving in rotation (4, 5) the stirring means at a variable speed.