EP1301586A1

EP1301586A1 - Cell culture chamber and bioreactor for extracorporeal culture of animal cells

Info

Publication number: EP1301586A1
Application number: EP01956613A
Authority: EP
Inventors: Jérôme Vetillard; Francis; Herodin; Richard Caterini
Original assignee: Gemcell Ltd (Societe de Droit Irlandais)
Current assignee: Gemcell Ltd (Societe de Droit Irlandais)
Priority date: 2000-07-19
Filing date: 2001-07-19
Publication date: 2003-04-16
Also published as: US20040132175A1; ATE275625T1; WO2002006441A1; JP2004504023A; EP1174497A1; EP1174497B1; CN1460122A; AU2001278542A1; CN1258588C; DE60013585T2; CA2416301A1; DE60013585D1

Abstract

The invention concerns a cell culture chamber having at least two planar filtering membranes with different cut-off, delimited by an envelope with axis of symmetry formed by an outer lateral wall, two end walls and inlet ports and outlet ports for dynamic liquid media, and a bioreactor containing the culture chamber for extracorporeal culture of animal cells.

Description

Cell culture chamber and bioreactor for the extracorporeal culture of animal cells

Field of the invention

The present invention relates to a cell culture chamber and a bioreactor containing it for the extracorporeal culture of animal cells.

The invention relates more particularly to a cell culture chamber with at least two flat filtering membranes with different cut-off threshold delimited by an envelope with axis of symmetry and a bioreactor which allows the cultivation of animal cells in said culture chamber while regulating and controlling the environment in which the cells are grown.

The cell culture chamber and the bioreactor of the invention for the extracorporeal culture of animal cells allow the culture of animal cells under sterile conditions. The invention applies to the production of animal cells such as hematopoietic cells, hepatic cells, skin cells (called keratinocytes), pancreatic cells, nerve cells organized or not in tissue structure in a therapeutic goal.

State of the art

As organ, tissue or cell transplantation represents a considerable medical technological advance, a keen economic interest has developed - consequently developed for the production of bioreactors intended for the culture of animal cells for a therapeutic purpose either by grafting said cells , or by using them in external devices that interface with the patient as palliative means for organic deficiency.

A major drawback of current bioreactors intended for the culture of eukaryotic cells lies in mass transfer, that is to say the mass transfer of nutrients and dissolved oxygen to the cells. eukaryotes, because these cells are fragile and are destroyed by mechanical stress generated by the agitation of the medium to aerate it.

US Patent No. 6,048,721 describes a bioreactor for the ex vivo growth and maintenance of mammalian cells. In this bioreactor, the discoid culture chamber is delimited by a planar bed of cells and a membrane permeable to gases and impermeable to liquid. The feed medium perfused in the lower compartment of the bioreactor diffuses radially and the air blown into the upper compartment oxygenates the medium. According to the mode of operation of this bioreactor, the medium responsible for the waste generated by the culture of the cells leaves in the sewer. The recovery of the cells after culture is done according to an enzymatic treatment. In this type of device, the thickness of the culture chamber risks inducing a partial oxygen pressure gradient which is poorly suited to good cell viability. The disadvantage of bioreactors known from the prior art and intended for the culture of eukaryotic cells lies in the fact that these bioreactors operate in continuous perfusion of their culture chamber, the flow of the nourishing medium is not recovered and is directly discharged to the sewer significantly increasing the cost price of culture.

However, for the culture of animal cells, such as for example hematopoietic cells or hepatic cells, a feed stream rich in growth factors proves essential to allow the multiplication and differentiation of said cells. Said growth factors being extremely expensive, the operation of a bioreactor which does not make it possible to recycle these factors represents a considerable financial cost, incompatible in the perspective of clinical exploitation.

The object of the present invention is to remedy all these drawbacks. O jet of the invention

The purpose of the present invention is to create ^a culture chamber and a bioreactor for extracorporeal culture of animal cells to maintain homeostasis of the surrounding environment and the cultured cells and allow them to grow in the best possible conditions.

To achieve this object, the invention develops the objectives set out below. An object of the invention is to maintain good cell viability within said culture chamber and of the bioreactor, and this by providing on the one hand to the cells of the culture medium a sufficient nutritional supply and by evacuating other apart from the wastes and inhibitors generated to allow growth of the cell population.

Another object of the invention is to be able to recycle the growth factors from the medium while evacuating sufficient cell waste from the culture medium and thus to achieve the economic optimization of cell culture.

Another object of the invention is to be able to carry out a targeted gene transfer on the cultured cells. Another object of the invention is to maintain the physico-chemical properties of the cell culture medium despite the disturbance induced by cell growth.

Another object of the present invention is to guarantee sterility, asepsis of the culture chamber and of the bioreactor, and in particular throughout the duration of cell culture.

Another object of the invention is to recover the cells cultivated within the culture chamber and the bioreactor of the invention.

Summary of the invention

The present invention is based on the observation that a culture chamber and a bioreactor for the Extracorporeal culture of animal cells could remedy the various drawbacks mentioned above if they allowed both to maintain good cell viability of the cultured cells while recycling the growth factors of the medium, thus ensuring a good level of cell proliferation.

Thus, the subject of the invention is a culture chamber for the extracorporeal culture of animal cells, delimited by an envelope with an axis of symmetry which is formed by an external side wall, two end and inlet walls and exits from dynamic liquid media, this chamber being characterized by the fact that it comprises: a) at least two flat filter membranes with different cutoff threshold, perpendicular to the axis of symmetry; b) between the membranes, a means forming a biocompatible culture support allowing adhesion of the cells in the culture state; c) two end walls constituting means for distributing dynamic liquid media; d) three pairs of inputs and outputs of dynamic liquid media (FI, F2, F3), intended to supply the cell culture chamber and selectively extract the cultured cells, the waste resulting from their culture and the excess nutrients , two of the pairs being, for each of them, connected between one of the end walls and one of the membranes, the third being connected between the two flat filter membranes.

The invention also relates to a bioreactor for the extracorporeal culture of animal cells comprising a culture chamber, delimited by an envelope with axis of symmetry formed by an external side wall, two end and inlet walls and exits from dynamic liquid media and comprising means for circulating said media in said chamber, this bioreactor being characterized in that it comprises: a) a culture chamber of said cells comprising at least two flat filtering membranes with different cutoff threshold, perpendicular to the axis of symmetry and that between said membranes with different cutoff threshold is located a means forming a biocompatible culture support allowing adhesion of the cells in culture state, said chamber being delimited by an envelope with axis of symmetry comprising two end walls constituting means for distributing the dynamic liquid media and three pairs of inputs and outputs of the dynamic liquid media FI , F2, F3, intended to supply the culture chamber of the cells and to selectively extract the cultured cells, the waste resulting from their culture and the excess nutrients, two of the pairs of which are, for each of them, connected between the 'one of the end walls and one of the membranes, the third being connected between the two filter membranes; b) means for circulating the first dynamic liquid medium FI according to a closed loop circuit and an expansion vessel RI containing said medium, these means being connected to said culture chamber; c) means for circulating the second dynamic liquid medium F2 in a closed loop or open loop circuit according to the function assigned to said medium and a reservoir R2 containing said medium, these means being connected to said culture chamber; d) means for circulating the third dynamic liquid medium F3 according to an open loop circuit and a reservoir R3 containing said medium, these means being connected to said culture chamber; e) control means for regulating and conditioning dynamic liquid media, connected to a regulation-command block.

The invention will be better understood thanks to a nonlimiting illustrative description of the culture and the bioreactor comprising said chamber by means of the following figures:

- Figure 1 shows a schematic view of a cell culture chamber delimited by an envelope with an axis of symmetry of hexagonal shape. FIG. 2 schematically represents the dynamic circulation of the liquid media FI and F3 for a pressure gradient pl> p3 in “downward phase” within the culture chamber of a bioreactor of the invention, in which the means forming the support biocompatible culture between the two membranes with different cut-off threshold is a bed of macro-supports. FIG. 3 schematically represents the dynamic circulation of the liquid media FI and F3 for a pressure gradient p3> pl in "ascending phase" within the culture chamber of a bioreactor of the invention, in which the means forming a support of biocompatible culture between the two membranes with different cutoff threshold is a bed of macro-supports.

FIG. 4 schematically represents a pressure gradient pl> p3 in “downward phase” within the chamber of a bioreactor.

FIG. 5 schematically represents a pressure gradient p3> p1 in "ascending phase" within the culture chamber of a bioreactor according to the invention. - Figure 6 shows a circuit diagram of a bioreactor where the regulation-control block is not shown.

FIG. 7 schematically represents the dynamic circulation of the liquid media FI and F3 for a pressure gradient pl> p3 in "downward phase" within the culture chamber of a bioreactor of the invention, in which the means forming the support biocompatible culture is a filter membrane, called culture. FIG. 8 schematically represents the dynamic circulation of the liquid media FI and F3 for a pressure gradient p3> pl in "ascending phase" within the culture chamber of a bioreactor of the invention, in which the means forming the support biocompatible culture is a filter membrane, called culture.

Detailed description of the invention

The invention relates to a cell culture chamber with an axis of symmetry, containing both the cells and the culture medium comprising at least two filter membranes with different cutoff threshold and a means forming a biocompatible culture support placed between two of the membranes. filtering planes with different cutoff threshold, said chamber being delimited by an envelope with an axis of symmetry formed by an external side wall and two end walls.

The first membrane, known as the feed membrane, has a cut-off threshold chosen in the interval ranging from 0.01 μm to 7 μm which allows biochemical exchanges within the culture chamber by allowing the passage of molecules from the nourishing medium such as the proteins and macromolecules, while achieving cell confinement preventing the cultured cells from leaving the homeostasis zone and also preventing the passage of contaminating particles by serving in particular as a barrier to bacteria which can contaminate said chamber.

Another filter membrane, called dialysis membrane, has a cutoff threshold of at most 15 KiloDalton

(KDa) and authorizes the molecular confinement of all molecules having a molecular mass greater than 15 KDa. The presence of this filtering membrane within the culture chamber makes it possible to define, with the first membrane, a confinement space for the culture cells. Consequently, the flat filtering membrane with cutoff threshold of at most 15 KDa confines the cells in culture in the culture chamber, as well as growth factors and large proteins.

According to a characteristic of the invention, at least

One of the membranes has a cutoff threshold preferably between 0.2 μm and 4 μm and the other a cutoff threshold preferably between 10 and

12 KDa.

According to the invention, the number of membranes present in the culture chamber can be greater than two. In this case, the additional membranes have cutoff thresholds adapted to those of the two aforementioned membranes.

According to the invention, the flat filter membranes with different cut-off thresholds within the

. cell culture are arranged perpendicularly to the axis of symmetry of said culture chamber.

According to the invention, the two flat filter membranes with different cutoff threshold can be mineral or organic membranes. By way of example, mention may be made of membranes made of biocompatible organic polymers, of cellulose or of polysulfone.

In addition, these two filter membranes are spaced from each other by at most about 25 mm and preferably by at most 20 mm, a distance which proves favorable for good development of the cells since they are almost always in contact with nutrient and oxygen sources.

Between the two filter membranes with different cutoff threshold is placed the means forming a biocompatible culture support allowing the adhesion of the cells in the culture state.

One of the means forming a biocompatible culture support, according to the invention, can be a bed of biocompatible macrosupports made up of particles of various sizes which can optionally be agglomerated into a continuous block by sintering of granular elements. This bed can have a thickness at most equal to the distance between the two filter membranes with different cutoff threshold. The said macrosupport bed plays on the one hand a role of supporting cells in a cultured state and on the other hand a mechanical role of maintaining the cell confinement space arranged between the two filter membranes with different cut-off threshold. According to the type of cells cultivated within the culture chamber of the invention, the type of biocompatible macrosupports will be chosen appropriately and of adequate size.

The macrosupports used between the two membranes of the culture chamber can have a cylindrical or spherical or even polyhedral shape such as for example massive machined blocks.

According to the invention, the macrosupports can be of mineral origin (such as, for example, coral), of metallic origin (such as titanium and its alloys for example) or also formed of biocompatible polymers.

By way of illustration, in the case of a culture of hematopoietic cells with a view to an application for a bone marrow transplant on the one hand and in the case of a culture of osteoblasts for the purpose of bone reconstruction on the other hand apart, the macrosupports can be coral microbeads. Such coral beads of desired particle size turn out to be macrosupports suitable for the applications mentioned above due in particular to their ability to be colonized by hematopoietic progenitors. In the case of bone precursors, the coral beads can also be metabolized, which would encourage their use in reconstructive surgery.

Whereas, for example, in the case of a culture of hepatic cells with a view to an ex-vivo blood detoxification application of a hepato-insufficient patient by an extracorporeal biomass of hepatocytes, the most suitable macro-supports may be polyamide beads, for example nylon®, fluorinated polymer beads, for example teflon®.

Thus, the presence of macrosupports between said membranes and the fact that the filtering membrane with cutoff threshold of at most 15 KDa does not allow the passage of cells in culture as well as that of the factors of growth, allow the confinement of cells in culture, which adhere to the so-called biocompatible macrosupports, in this cell confinement space located between the two membranes with different cut-off threshold.

Another means forming a biocompatible culture support according to the invention may be a so-called M2 culture filter membrane having specific characteristics distinguishing it from the other membranes mentioned above, that is to say the so-called feed membrane filter with threshold cutoff in the range from 0.01 μm to 7 μm and the so-called dialysis membrane with cutoff threshold of at most 15 KDa.

This culture membrane, placed between the two aforementioned membranes, can rest on the so-called dialysis membrane with a cutoff threshold of at most 15 KDa.

Said culture membrane can be a membrane of mineral or organic origin, the composition of which can vary according to the different types of culture and the culture conditions.

Thus, the so-called culture membrane, on which the culture cells can multiply, can be modified by grafting of substrates or by cell co-cultures.

As nonlimiting illustrative examples, various types of modifications can be mentioned, which are:

- the binding of ligands for adhesion molecules of glycoprotein types,

- or the binding of antibodies, - or the formation of a substratum from a first cell type which constitutes the first culture of adherent cells, operated in another bioreactor or in conventional culture, then after transfer to the culture chamber of said substrate, establishment of a second cell type, and optimization of coculture conditions. However, the formation of said substratum can also be done in the culture chamber according to the invention from a first cell type, followed by rinsing said substrate, the establishment of a second cell type and the optimization co-cultivation conditions.

- or the fixation of protein molecules.

The culture membrane which is also filtering has a cutoff threshold chosen in the range from 0.01 μm to 7 μm.

In the case of the implementation of such a so-called culture membrane as a means forming a biocompatible culture support according to the invention, placed between the so-called feed membrane filters with cut-off threshold chosen in the range from 0, 01 μm to 7 μm and called dialysis with cut-off threshold of at most 15 Kda, these last two membranes can be supported by appropriate mesh supports allowing the dynamic liquid media FI, F2, and F3 intended to supply the chamber to pass through. cells and selectively extract the cultured cells, the waste resulting from their culture and the excess nutrients.

According to the need, said mesh supports intended to support the aforementioned membranes can be placed in contact with one or the other face, or both sides of said membranes, as well as in contact with one and / or the other side of the culture membrane. The cell culture chamber as previously mentioned comprises an envelope with an axis of symmetry formed by an external side wall and by two end walls which can be likened to two flat bottoms situated at each of the ends of said side wall.

This envelope with an axis of symmetry may consist for example of a biocompatible polymeric material or of stainless steel. Mention may be made, as biocompatible polymeric materials, of polyolefins, polyamides, polyesters, or fluorinated polymers and others.

The supply of the culture chamber with dynamic liquid media is carried out in a homogeneous manner due to the excellence of the distribution by the internal faces of the end walls of the envelope and the distribution of said media within said chamber. through entrances and exits from these judiciously placed environments.

According to the invention, the internal faces of the two end walls constitute means for distributing dynamic liquid media.

According to a first type, the internal faces of the end walls of the envelope with an axis of symmetry are smooth, so that the distribution of the dynamic liquid media supplying the culture chamber in contact with the supply membranes and of dyalise, can be carried out in a homogeneous manner and naturally without constraint.

According to a second type, which ensures an organized supply of the culture chamber, in liquid supply media, in contact with the supply and dialysis membranes, the internal faces of the two end walls of the envelope with axis of symmetry are provided with distribution streaks for supplying said culture chamber with two of the three media dynamic liquids. These distribution streaks constitute a main network of so-called main streaks. This main network of ridges located on the internal face of each of the two end walls, facing the cell culture space, can be divergent from the inlet tubing arranged in said wall.

According to the invention, the main network of said main streaks, also called distribution network, allows a homogeneous distribution of the two dynamic liquid media, which are conveyed to the culture chamber by means of biocompatible conduits, the ends of which are connected at the end walls. The number of distribution streaks located on the face opposite the cell culture space of each of the two end walls is defined so as to obtain a good dispersion of the dynamic liquid medium in the culture chamber and will be determined as a function of the shape of the envelope with axis of symmetry Said main network is supplemented by a secondary network, formed of so-called secondary streaks, shallower and of direction substantially orthogonal to the main network so as to promote circulation between the distribution areas delimited by the main network. Thus, the secondary streaks of the secondary network are substantially perpendicular to the streaks of the main network. This secondary network can see its spacing vary so that the secondary streaks are closer and more numerous on the side opposite the entry of the medium flow to facilitate the drainage and evacuation of said medium.

Two adjacent streaks in the main network constitute a distribution area and two adjacent streaks in the main network intersected by two adjacent streaks in the secondary network constitute a distribution cell.

The main and secondary networks form a fine "grid" network for the propagation of dynamic liquid media. All of these two networks can by example form a mesh network of the waffle type.

The main network being located on the opposite face of the cell culture space, has a certain height and a certain width that the skilled person is quite able to define, knowing that the network of streaks must be in contact with the flat filtering membrane to ensure the cohesion of the assembly. In fact, the volume formed by the assembly integral with the network of streaks and the membrane forms the grid for distributing the fluid flow which can represent approximately 50% of the surface of the membrane.

Thus, for example, the main network may consist of main streaks having a depth of at most 5 mm and a width of at most 2 mm, the pitch between two adjacent streaks of said main network may be at most 2 mm. Similarly, the secondary network may consist of secondary streaks having a depth of at most 2 mm and a width of at most 2 mm, the pitch between two adjacent streaks of said network gradually decreasing from the entry side of the flow. medium to the outlet of said medium flow. Therefore, in its distal part - that is to say on the side of the outlet from the middle - the pitch of the secondary network will be at most 2 mm.

Consequently, the end walls can be envisaged as producing a fine pattern of cells, for example of the waffle type on which the membranes with different cut-off threshold come to bear or are glued with a biocompatible adhesive of the polymer adhesive type.

As for the envelope with an axis of symmetry of the culture chamber, it is formed by an external side wall and two end walls.

The external side wall can be formed of at least three sections of the same section, each having an adequate height, which can be identical. These at least three wall sections constitute the external walls of at least at least three stackable modules (Cl, C2, C3), two of which

(C1 and C3) receive the end walls of the envelope with axis of symmetry of the culture chamber, the third module (C2), inserted between the two preceding ones, receiving at one of its ends the flat filtering membrane (Ml) said supply with cut-off threshold included in the range from 0.01 μm to 7 μm and its other end, the flat filtering membrane called dialysis with cut-off threshold of at most 15 KDa.

These at least three modules are superimposable and are connected to each other in a leaktight manner by means of seals, by an appropriate fixing means, such as, for example, by gluing, mechanical mounting by screws or the like.

The shape of the envelope with axis of symmetry of the culture chamber according to the invention can be chosen appropriately to facilitate, for example, a stacking of several culture chambers on a support. Those skilled in the art are able to choose the shape of the envelope with axis of symmetry of said culture chamber according to the use that will be made of it. By way of example, it is possible to envisage an envelope with an axis of symmetry, with a section of circular or polygonal shape.

It should be emphasized that a stack of modules for culture chambers can be produced provided that said modules have the same shape in order to obtain a stable stack of these modules on an adequate support. For example, according to an embodiment of a culture chamber by stacking several modules, a hexagonal shape for example can facilitate the successive stacking of these modules on an appropriate base having for example six columns. In this case, the six columns of the base playing the role of support for the stacking of these modules can also serve for example as supply and evacuation conduits for dynamic liquid media. As for the supply of dynamic liquid media to the culture chamber according to the invention, it is carried out by a system with three dynamic liquid media, namely a system with three separate media flows (FIGS. 1 to 8).

A first dynamic liquid medium (FI), entering and leaving through biocompatible tubes connected to the side wall of the module (Cl) close to one of the end walls of the envelope with axis of symmetry (for example the wall of upper end) supplies the culture medium with nutritive medium (also called rich nourishing medium), this medium being rich in growth factors. This first dynamic liquid medium is composed of elements necessary for the culture of cells such as for example proteins, trace elements, glucose, water and growth factors, and supplies the culture medium in a nutritional medium. fresh.

A second dynamic liquid medium (F2) entering and leaving via biocompatible tubing connected to the external lateral wall of the module (C2) forming part of the envelope with axis of symmetry of the culture chamber can have three distinct functions depending on the uses that will be made of it.

According to a first function, this second dynamic liquid medium (F2), entering via a biocompatible tubing connected at the level of the side wall of the culture chamber of the module (C2), serves to introduce into said module of said chamber the cells intended for be cultured and recover said cultured cells within said chamber module after their cultivation (hematopoietic cells or hepatic cells for example). According to a second function, this second dynamic liquid medium can play the role of gene transfer. In fact, during the introduction of the culture cells in suspension in the module (C2) of the culture chamber via this second liquid medium dynamic, viral particles contained in said liquid medium can attach to cells in suspension at their membranes and thus allow the desired gene transfer. This second flow of medium can make it possible to obtain the desired culture cells, genetically modified, which it is desired to cultivate. This medium in fact transports the gene transfer vectors and allows them to be brought into contact with the cells in order to establish a membrane fusion between the target cell and the gene transfer vector. These gene transfer vectors are of all kinds. By way of illustration, there may be mentioned viruses such as for example adenoviruses, retroviruses, liposomes, plasmid complexes. Such labeling of cells (by the injection of gene transfer vectors) carried out outside the human body can make it possible to precisely target the tissue to be genetically modified. In addition, a synthetic disposable virus can be used, for example, to target gene transfer to a cell population of therapeutic interest. This virus characterized in that the genes coding for the envelope and those which carry the genetic information strictly speaking are separated, is incapable of reproducing inside the cells afterwards _. have transferred the desired genetic information. Thus the risks of genesis of a recombinant virus from the synthetic virus used and from a wild virus which could be present in the patient are limited.

According to a third function, this second dynamic liquid medium (F2) can play the role of flushing flow of inhibitory macromolecules present at the level of the cell confinement space. In fact, the cells placed in culture may have undergone stress before their harvesting or during their pre-inocular treatment or even during their inoculation in the culture chamber. This stress can induce the production (by said cells) of proteins which will inhibit their capacity to multiply by causing them to go into a dormant state, called quiescent state. During this state, said cells are no longer in a physiological state to respond to stimulation by growth factors, by multiplying.

In the case of hematopoietic cells, mention may be made of "radio-induced" stress which is caused by exposure to ionizing radiation (volunteers for radiotherapeutic or accidental use) and is subsequently manifested by stress. This type of stress leads to the production of inhibitors such as, for example, "Transforming Growth Factor-beta" or "Tumor Necrosis Factor-alpha". These inhibitory molecules being cytokines in the same way as the growth factors provided for the culture of hematopoietic cells, they are therefore confined by the membranes with a different cut-off threshold from the culture chamber.

To carry out an appropriate culture of the cells, these inhibitory molecules must be removed from the culture medium and therefore from the culture chamber. The second dynamic liquid medium in its third function therefore serves to rinse the cell confinement zone to remove said inhibitory molecules.

A third dynamic liquid medium (F3), entering and leaving through biocompatible tubes connected to the side wall of the module (C3) close to the second of the end walls of the envelope with axis of symmetry (for example the wall of lower end) supplies the culture medium with a basic nutritive medium (also called basic-regenerative nutrient medium). This basic nutritive medium is a nourishing medium totally devoid of growth factors. Such a basic medium is therefore composed of glucose, water, trace elements such as for example vitamins and minerals, dyes for evaluating the pH of the medium and proteins of the albumin type.

According to the invention, this culture chamber intended to be supplied by the system with three distinct dynamic liquid media (FI, F2, F3) (also called triple flow) has the characteristic of three pairs of inputs and outputs of said media. Two of these pairs (FI, F3) are connected close to the end walls to supply the modules (Cl) and (C3) and allow their distribution in contact with the flat filter membranes with different cutoff threshold. The third pair (F2) is connected to the external side wall at a level located between the two flat filter membranes with different cutoff threshold, that is to say connected to the module (C2).

The offset arrangement of these pairs of media inlets and outlets on the envelope delimiting the culture chamber makes it possible to obtain a homogeneous distribution of said liquid media within said chamber (see FIG. 1).

According to the characteristics of the invention, the inlet and outlet pipes of the triple flow system are therefore distributed appropriately over the envelope with axis of symmetry of the culture chamber. In accordance with FIG. 1, the biocompatible inlet tubing of the first flow denoted EF1 is connected at a level close to the upper end wall of the envelope of the culture chamber, while the outlet tubing denoted SF1 of the first flow is also connected on the same end wall but opposite the inlet pipe.

The biocompatible inlet tubing of the third flow, denoted EF3, is connected to a level of the lower end wall of said casing so that this tubing is connected at an angle of approximately 120 ° relative to the tubing. inlet of the first feed stream (EF1) rich in growth factors, to allow good distribution of said media within the culture chamber. Furthermore, the biocompatible outlet tubing for this third stream, denoted SF3, is also connected to the same end wall but opposite the inlet tubing for said stream (EF3). In accordance with FIG. 1, the biocompatible inlet tubing of the second dynamic liquid medium denoted EF2 is connected between the two flat filter membranes denoted (Ml) and (M3), which respectively have a cutoff threshold of 0.22 μm and of 10 KDa, at the level of the external lateral wall of the envelope at an angle of approximately 60 ° relative to the inlet tubing of the first flow (EF1). The biocompatible outlet tubing denoted SF2 is connected opposite to the inlet tubing of said flow on the side wall.

Consequently, the three pairs of inputs and outputs of dynamic liquid media are placed in three vertical planes passing through the axis of symmetry of the envelope, these planes being offset by an angle of about 60 ° between the first inlet and the second inlet, and by an angle of approximately 120 ° between the first inlet and the third inlet of the dynamic liquid media, the outputs of said liquid media being in the same angular arrangements.

The present invention also relates to a bioreactor comprising the cell culture chamber previously described. This culture chamber, by means of these three pairs of inputs and outputs of dynamic liquid media, is connected by. connecting conduits to supply tanks and / or evacuation tanks of said chamber. The bioreactor according to the invention furthermore comprises means for regulating the culture conditions and for controlling mass transfer of said dynamic liquid media, connected to the regulation-command block of said bioreactor.

Thus, this bioreactor comprises, in addition to the culture chamber, “a regulation block also called control block” which allows the control and regulation of the pH, of the oxygen concentration and of the temperature of the culture medium. The “regulation-command block” can automatically manage the complete operation of the bioreactor. This regulation is of the P.I.D. type. Digital

(proportional, integral and Derivative) and the data returned by the various probes can be recorded and analyzed by computer.

Therefore, the bioreactor of the invention containing the culture chamber mentioned above, is organized into interchangeable functional unit modules sized for a certain value of mass or energy transfer (such as, for example, aeration modules , heat exchangers), constituting a modular unit resizable by juxtaposition of identical functional units in series and / or in parallel, and is provided with a programmable regulation-command block.

The regulation-command block receives all the information relating to the dynamic liquid media FI, F2, F3, by the control and regulation means, as well as the information relating to the various tanks, pumps, valves and pressures prevailing in the zones. -Cl, C2, C3 modules of the culture chamber, manages them and issues the necessary operating orders.

Therefore, it is possible to modify on demand the internal characteristics of the culture chamber and to change the parameters and regulatory programs contributing to the homeostasis of the medium in order to adapt to the cell type cultivated.

The regulation assembly can also have a crystal infrared spectrum measurement cell, which allows by deconvolution of the re-emission spectrum to measure the concentration of certain solutes in the culture medium. It is then possible to follow “online” and “in real time” the glucose and lactate concentrations.

Thus, the temperature of the culture medium can be fixed at a set value between 30 and 38 ° C. During cell growth, the pH of the culture medium can be fixed at a set value between 6.5 and 7.7. The regulation-control block also makes it possible to regulate the air flow and the rate of C0 ₂ which by its dissolution gives the amphoteric HC0 ₃ ^~ which buffers the medium and contributes to the stability of the pH.

According to one of the characteristics of the bioreactor of the invention, the inlet and outlet pipes (of the chamber) of the first dynamic liquid medium FI are connected by connecting pipes to a reservoir RI of nourishing medium rich in growth in a closed loop circuit allowing the recycling of the growth factors necessary for the development of cells in culture. This RI tank can act as an expansion tank.

According to one of the characteristics of this bioreactor, said connecting conduit between the inlet tubing of the first rich nourishing medium FI of the chamber and said tank denoted RI of this medium is equipped with a pump PI making it possible to control and adjust the volume and the flow rate of the FI medium circulating in a closed circuit in the culture chamber.

The closed loop conveying the rich feed stream FI has an air vent which is located on the expansion vessel RI, this air vent is provided with a cut-off filter of 0.22 μm guaranteeing middle asepsis. Likewise, this loop is provided with a solenoid valve VI controlled by the programmable regulation-control block allowing pressure balancing with atmospheric pressure on demand. The RI expansion vessel is also fitted with high and low level sensors for liquid medium used to trigger the reversal of the circulation of fluids. According to another characteristic of the bioreactor of the invention, the inlet tubing of the third basic feed medium (EF3) of the culture chamber is connected by a connecting duct to a reservoir denoted R3 of this medium, and the tubing of outlet (SF3) is connected by a conduit to the sewer (waste recovery compartment).

According to the invention, the connecting pipe connected to the inlet pipe of the third dynamic liquid medium EF3 mentioned above is equipped with a pump P3 then that the conduit connected to the outlet tubing of said flow is equipped with a valve V3 possibly controlled by the regulation-command block, the assembly making it possible to adjust and control the volume and the flow rate of said third medium F3 circulating in open circuit in the culture chamber.

According to the invention, a device for aerating the rich feed medium FI and a device for aerating the basic feed medium F3 are provided on one and the other of the two circuits in closed and open loops placed respectively between the inputs of said dynamic liquid media FI and F3 in the cell culture chamber and the Ri and R3 reservoirs.

According to the invention, a heat exchanger for the rich feed medium FI and a heat exchanger for the base feed medium F3 are provided on each of the two circuits at the inputs of said dynamic liquid media FI and F3 into the cell culture chamber.

These devices make it possible to maintain the homeostasis of the culture chamber by preconditioning the flows of media which penetrate therein so as not to bring into the culture chamber an element of volume of culture medium which can induce a brutal disturbance of its physical parameters. -chimiques. According to another characteristic of the bioreactor of the invention, the inlet tubing of the second dynamic liquid medium EF2 of the culture chamber, located at the side wall of the envelope, is connected by a connecting duct to a reservoir noted R2 containing the second dynamic liquid medium chosen as a function of the role assigned to it, namely either supplying the culture chamber with cells intended to be cultured and discharging said chamber after culture, either carrying out a gene transfer, or having a role of flushing flow intended to discharge the culture chamber of the inhibitory molecules from the cells to be cultured.

Depending on the function assigned to the second dynamic liquid medium of the culture chamber, the tubing of SF2 outlet, located opposite the EF2 inlet tubing on said wall, will be connected: by a connecting duct to a cell recovery tank after culture (i.e. a collection bag) or to the sewer ( compartment for the elimination of inhibitory molecules) according to an open loop circuit or via a connection conduit to a reservoir containing the medium provided with suitable vectors for gene transfer according to a closed loop circuit.

According to another characteristic of the bioreactor of the invention, one or more functional medium exchanger modules, also called dialyzers or ultrafiltrators, can be added to purify the feed medium FI at the outlet of the culture chamber. This or these medium-medium exchange functional modules are located outside the culture chamber, mounted in series on the outlet connection duct of the closed-loop circuit of the first feed medium FI rich in growth factors and traversed against the current. by the basic feeding medium F3 in open loop.

In the case of the use of two functional modules connected in series ad hoc on the closed loop FI, it is possible to use two different cutoff thresholds of, for example, 10 KDa and 30 KDa. We can, by injecting the flux from the 10 KDa module into the 30 KDa module, and recovering the permeate - that is to say the liquid fraction which has passed through the membrane at 30 KDa cutoff threshold to reinject it into the IF loop, carry out a windowing of the proteins of the medium between 10 and 30 KDa which corresponds to the size of the growth factors. Such additional functional modules can prove useful when, for example, the growth factors are produced by support cells in a first annex culture chamber, and when it is desired to condition this medium (that is to say recover the growth factors produced, without recovering the cellular waste generated by these support cells) in order to be able to reuse it in the IF loop in order to stimulate the cells said to be of therapeutic interest present in the main culture chamber.

It should be noted that the method used to cultivate hematopoietic and / or blood cells can use support cells called .stromal cells. These stromal cells can be genetically engineered to produce human cytokines at expression levels such that they can partially meet the cytokine requirements of the culture.

In addition, it should be emphasized that the envelope with axis of symmetry making it possible to obtain a homogeneity of distribution of the nutrients with an optimal dispersion has the advantage of being sterile in the same way as all the elements of the bioreactor in contact with the cells and the culture medium. In fact, the sterilization of the bioreactor is carried out by autoclaving at 121 ° C. for 20 minutes of the entire apparatus as well as the bottles of reservoirs. The connecting conduits, tanks and other sealing elements are made of biocompatible materials which can withstand ten sterilization cycles without damage in the case of laboratory use. On the other hand, the whole culture chamber, connection conduits, media reservoirs and the like will constitute a single-use culture kit in the case of use in human clinic. Furthermore, the reconditioning of the bioreactor after cell culture, within the framework of a laboratory type use, is done by protein digestion with a molar hydrochloric acid solution followed by an ultra pure rinsing, a sterilization.

According to the operating mode of the bioreactor of the invention and in accordance with FIGS. 2 and 7, the rich nourishing medium FI is introduced at the level of the upper end wall of the envelope in the zone (Cl) (module or compartment Cl) of the culture chamber between said wall and the flat filtering membrane with cut-off threshold of the order of 0.01 μm to 7 μm, by opening the PI pump located on the connection connecting the culture chamber to the reservoir RI containing said medium FI while the pump P3 located on the connection conduit connecting the culture chamber to the reservoir R3 containing the basic nourishing medium F3 is stopped. In addition, the pressure p3 prevailing in the zone (C3) (module or compartment C3) of the culture chamber situated between the lower end wall of the envelope and the flat filtering membrane with cutoff threshold of at most 15 KDa, is adjusted by the back-pressure valve denoted V3 located on the conduit connecting the culture chamber to the sewer so that the pressure pi prevailing in the zone Cl of the culture chamber is greater than the pressure p3 and that the pressure p2 prevailing in the zone (C2) (module or compartment C2) is between pi and p3 according to a pressure gradient.

Thus, during the introduction of the first dynamic liquid medium FI into the culture chamber by the opening of the pump PI, the growth factors of the nutritive medium pass into the zone denoted Cl and through said membrane with cut-off threshold of the order of 0.01 μm to 7 μm from the culture chamber, but are retained by the flat filtering membrane with a cutoff threshold of at most 15 KDa, which acts as a barrier for the passage of growth factors and big proteins.

Therefore, the. growth factors can migrate on either side of the flat filtering membrane with cut-off threshold of the order of 0.01 μm to 7 μm ensuring their role of stimulating growth and / or controlling differentiation for cells in a culture state confined between the two flat membranes of the culture chamber defining the zone C2 (module or compartment C2) of said chamber. In accordance with FIG. 4, the flat filtering membrane M3 with a cutoff threshold of at most 15 KDa confines the growth factors and the large proteins of the fresh nutritive medium F1 in zones C1 and C2 of the culture chamber. On the other hand, the trace elements of said nutritive medium F1 and the waste of small sizes (such as, for example, NO, NH ⁺ , lactate and others) generated by the culture of the cells confined in the zone C2 of said chamber, are drained to zone C3 where they are taken to the sewer. The overall transmembrane flow is therefore oriented from zone C1 towards zone C3 of the culture chamber (see FIGS. 2 and 7).

In this operating mode of the bioreactor according to the invention, a mode called “downward phase”, the fresh nutritive medium F1 circulating in a closed loop loses volume due to the drainage of the aqueous solution towards zone 03 and in particular towards the sewer. . Consequently, the volume of feed medium F1 in the reservoir RI decreases. The growth factors and the large proteins of the fresh nutritive medium F1 being confined in zones C1 and C2, the waste is purged from the zones of the culture chamber towards zone 03 and is drained towards the sewer by means of the valve V3 opened.

As soon as the volume of the reservoir Ri or expansion vessel of the closed loop carrying the fresh nutritive medium Fl reaches a certain low level, the regulation-command block of the bioreactor programmed according to a particular sequence reverses the flow of flow. As a result, the pump PI which was running goes to stop and the pump P3 to stop starts up. And, the open valve V3 is then closed.

Consequently, the pressure pi prevailing in the zone C1 of the culture chamber becomes lower than the pressure p3, while the pressure p2 prevailing in the zone 02 is between pi and p3, according to a pressure gradient inverted compared to the mode from previous operation. The overall transmembrane flow is then oriented from zone C3 towards zone C2 of the culture chamber (see FIGS. 3 and 8). In this mode of functioning of the bioreactor and in accordance with FIGS. 3, 8 and 5, mode called “ascending phase”, the reversal of flow within the culture chamber allows the fresh nutritive medium F1 circulating in the culture chamber to be recharged with nutrients costs from the third dynamic liquid medium F3 and to compensate for the losses caused, inter alia water, by the "downward phase". This basic (regenerating) F3 medium replenishes the cell culture chamber with glucose, water and trace elements.

As soon as the volume of the reservoir RI or expansion tank of the closed loop circuit conveying the fresh nutritive medium F1 reaches a certain high level, the control system of the bioreactor, programmed according to a particular sequence again reverses the flow circulation.

Thus, the growth factors due to the closed-loop circulation of the rich nourishing medium F1, that is to say of the first dynamic liquid medium of which they are a part, are confined in the closed loop and in the zones C1 and C2. , and their concentration oscillates between a concentration C ₀ and Co +/- ΔC. The rich culture medium is therefore recycled and the use of growth factors optimized thanks to the culture chamber and the bioreactor of the invention. This flow reversal system within the culture chamber makes it possible to create transmembrane flows having low hydrodynamic constraints compatible with the fragility of the cultured cells. A “laminar” slow flow system is thus obtained. This reversal of flows can also prevent clogging of filter membranes, the transmembrane pressure drop (index measuring the permeability of the membrane) can be monitored in real time by electronic manometers placed on each flow of dynamic liquid media.

In the operating mode of the bioreactor of the invention, the second dynamic liquid medium (F2) entering and leaving at the level of the external lateral wall, between the two flat filter membranes of the culture chamber, circulates within said chamber in an open loop or closed loop circuit according to the function assigned to it, the pumps PI and P3 being stopped, and its input and output configuration is fixed by the function you want to assign to it.

According to the operating mode of the bioreactor, when the second dynamic liquid medium F2 in its first function is used to inoculate the cells to be cultured in the culture chamber and to discharge them after culture, it functions in open loop in the same way as when it serves in its third function to rinse the chamber to remove the inhibitory molecules. On the other hand, when the dynamic liquid medium F2 has a role of gene transfer, it functions in a closed loop during the time of inoculation and incubation.

In its first function and according to the operating mode of the bioreactor in accordance with FIG. 6, the second dynamic liquid medium F2 containing the cells to be cultured is inoculated in zone C2 of the culture chamber by a syringe through a biocompatible septum positioned on one of the three branches of the inlet manifold EF2 (of the external side wall) whose solenoid valve V2E1 is open, the solenoid valves V2E2 and V2E3 being closed. During the inoculation of said medium, the three solenoid valves V2S1, V2S2 and V2S3 located on the three branches of the outlet manifold SF2 arranged opposite the inlet manifold EF2 are closed. During the recovery of the cells after culture, the two solenoid valves V2E2 and V2E3 of the inlet tubing EF2 are closed, the solenoid valve V2S1 located on one of the three branches of the outlet tubing SF2 connected to a conduit connecting to the recovery tank for cultured cells, is open while the solenoid valves V2S2 and V2S3 of the other two branches of the outlet pipe SF2 are closed. The regenerating base medium is then pumped by starting the pump P2 from the reservoir R2 and serves to purge the content of the compartment C2 (between the two membranes with different cutoff threshold) in the cell collection pocket. An enzymatic treatment of the trypsin and / or collagenase and / or DNAse type could be used to destructure the extracellular matrix produced by the cells during the culture to facilitate their anchoring. In the context of a culture of hematopoietic cells on a macrosupport of the coral type, a slight acidification of the medium at pH = 6.0 or pH = 6.5 will allow the coral to be dissolved and the hematopoietic cells to be recovered after rinsing.

In its third function and according to the operating mode of the bioreactor, the second dynamic liquid medium F2 containing the elements necessary for rinsing the cell confinement space operates in open loop according to the same principle of closing and opening of the solenoid valves as the operation described above except that the inlet tubing is not provided with a biocompatible septum but is connected by a connecting conduit to the reservoir R2 containing said chosen medium F2.

When the rinsing medium F2 is introduced into the zone C2 of the culture chamber at one of the three branches of the inlet tubing EF2 (of the external side wall), the solenoid valve V2E3 of which is open, the solenoid valves V2E1 and V2E2 are closed. During the introduction and recovery of said rinsing medium, the two solenoid valves V2S1, V2S2 located on the two branches of the outlet tubing SF2 are closed, the solenoid valve V2S3, located on the other branch of the outlet tubing SF2 being open, the rinsing flow circulating continuously in open loop. The outlet of the V2S3 solenoid valve can be connected to a pipe leading to the sewer. The sewer consists of a tank of sterilized medium at the same time as all the tubing of the bioreactor, its tanks, its functional modules and the culture chamber, which guarantees the sterility of the flow. Two 0.22 μm filters can be added to this sewer while increasing safety and ensuring sterility, - 3.1 - especially when the sewage tank must be changed "hot" after an overflow.

In its second function and according to the operating mode of the bioreactor in accordance with FIG. 6, the second dynamic liquid medium F2 containing the gene transfer vectors is introduced into the zone C2 of the culture chamber by the opening of the solenoid valve V2E2 located on one of the three branches of the inlet manifold EF2, the solenoid valves V2E1 and V2E3 being closed. During the introduction of said medium, the two solenoid valves V2S1, V2S3 located on two branches of the outlet pipe SF2 are closed. This F2 medium containing the gene transfer vectors circulates in a closed loop for the time necessary for inoculation and incubation. When the gene transfer procedure is complete, the F2 loop returns to rinsing mode as described above, in order to rinse any residual vectors. Then, the rinsing being finished, the culture continues according to the alternation of the "ascending" and "descending" phases of the flows F1 and F3.

It should be noted that one of the unwanted epiphenomena during the reversal of flow from the downward phase to the upward phase is the possibility that the medium F1 is slightly contaminated by .waste from zones C2 and C3. Now it turns out that the dilution phenomenon makes this possible contamination negligible. In addition, the proximal part of the connecting conduit connecting the base medium reservoir F3 and the culture chamber is charged with fresh F3 medium, that is to say from said reservoir, only the distal part of the conduit connecting the chamber sewer culture, that is to say near the valve V3, is loaded with waste.

The culture chamber and the bioreactor according to the invention can be used for the extracorporeal culture of animal cells and, as such, the invention relates to the medical field. As an illustration, the culture chamber and the bioreactor according to 1 invention can be applied to the production of hematopoietic cells or liver cells.

The present invention thus finds its application for the extracorporeal culture of bone marrow cells. Hematopoiesis is the physiological process that renews all the figured elements of the blood

(mature blood cells with a limited lifespan) through the multiplication and differentiation of an original multipotent primitive cell (called stem cell) capable of generating all cell types, blood cells also called blood figurative elements. This process is under the control of hematopoietic growth factors, also called cytokines. The transplant of immature hematopoietic cells, called hematopoietic progenitors, in a patient subjected to an accidental aplasia or following an anticancer treatment is a means of being able to revive the activity of the injured bone marrow to initiate a resumption of hematopoiesis.

This process implies that progenitors - cells at an early stage of development - are taken from the patient by cytapheresis or directly by puncture of the bone marrow, and are re-injected after culture so that these cells can replenish the patient's blood and immune system ( in the case of complete aplasia) or to compensate for the morbid period of transient pancytopenia (phase of neutropenia, lymphopenia and thrombocytopenia) in order to allow the endogenous haematological recovery of the aplastic victim (in the case of accidental irradiation).

Likewise during acute liver failure leading to a very short-term life-threatening risk, it is necessary to compensate for the patient's deficient hepatic functions by an extracorporeal blood detoxification treatment therefore requiring a large number of hepatocytes to perform the necessary metabolic functions. . The culture chamber as well as the bioreactor described above can allow the production of liver cells in sufficient number to obtain an effective treatment and rapidity of detoxification compatible with the hemodynamic constraints posed by the extracorporeal circulation of the patient's blood.

By way of illustration, a bioreactor with a useful volume which can vary from 50 to 100 ml makes it possible to carry out an extracorporeal culture of animal cells over a period of ten - days within the framework of the production of a cellular biomass to use of grafts and / or as a palliative for a deficiency. As part of the reconstruction of an organized and hierarchical tissue model, the culture times authorized by the bioreactor can reach several months.

For a bone marrow cell transplant (hematopoietic cells), it is necessary that 10 ^δ to 10 ⁷ CFU (type of immature cells) / kg of the patient's weight come from the culture. This clinical threshold, which partly determines the success of the transplant, makes it possible to estimate at 10 ¹⁰ the number of cells from hematopoietic culture necessary for the transplant. The culture chamber or chambers as well as the bioreactor as described can be sized for this type of application.

The conditions of pH, temperature and partial pressure of oxygen for a culture of the cells mentioned above will be of the order of 7.4 for a growth pH, of the order of 37.5 ° C for the temperature and a partial oxygen pressure (in% of saturation) equal to 15%. The nourishing medium is a synthetic medium consisting of ultra-purified or synthesized proteins and trace elements (such as iron, selenium, transferrin, vitamins and others) and a vital dye. This nourishing medium is enriched by growth factors which are cytokines whose concentration can vary from 10 to 100 ng / l according to the needs. In addition, cytokines can be stabilized in a biologically active state with the presence heparan. The basic nourishing medium -regenerating- is marketed under the following trade names, it can be MEM-alpha or RPMI1640 or even IMDM.

Claims

Culture chamber for the extracorporeal culture of animal cells, delimited by an envelope with axis of symmetry which is formed by an external side wall, two end walls and inlets and outlets of dynamic liquid media, characterized in that that it includes:

a) at least two flat filter membranes (Ml and M3) with different cutoff threshold, perpendicular to the axis of symmetry; b) between the membranes (M1 and M3) a means forming a biocompatible culture support allowing adhesion of the cells in the culture state; c) two end walls constituting means for distributing dynamic liquid media; d) three pairs of inputs and outputs of dynamic liquid media (F1, F2, F3), intended to supply the cell culture chamber and to selectively extract the cultured cells, the waste resulting from their culture and the excess nutrients , two of the pairs being for each of them connected between one of the end walls and one of the membranes, the third being connected between the two flat filter membranes.

Culture chamber according to claim 1, characterized in that one of the planar filter membranes has a cut-off threshold of between 0.01 μm and 7 μm, and the other planar filter membrane has a cut-off threshold of at most 15 KiloDalton (KDa).

Culture chamber according to either of claims 1 and 2, characterized in that one of the flat filter membranes has a cut-off threshold preferably between 0.2 μm and 4 μm and the other planar filtering membrane has a cutoff threshold preferably between 10 and 12 KDa.

4. Culture chamber according to at least one of claims 1 to 3, characterized in that the two membranes with different cut-off threshold are spaced from each other by at most 25 mm and preferably between 0.2 and 20 mm.

5. Culture chamber according to at least one of claims 1 to 4, characterized in that the means forming a biocompatible culture support allowing adhesion of the cells in the culture state is a bed of biocompatible macrosupports.

6. Culture chamber according to claim 5, characterized in that the macrosupport bed has a thickness at most equal to the distance between the two filter membranes with different cutoff threshold.

7. Culture chamber according to at least one of claims 5 and 6, characterized in that the macrosupports present between the membranes have a cylindrical, or polyhedral or spherical shape.

8. Culture chamber according to at least one of claims 5 to 7, characterized in that the material constituting the macrosupports is chosen from the group of mineral materials, metallic materials and biocompatible polymeric materials.

9. Culture chamber according to claim 8, characterized in that the material constituting the macrosupports is preferably chosen from the group consisting of coral, titanium and its alloys, polyamides, fluorinated polymers.

0. Culture chamber according to at least one of claims 1 to 4, characterized in that the means forming a biocompatibility culture support allowing the adhesion of the cells in the culture state is a filtering membrane for M2 culture placed between the two membranes Ml and M3 filtering planes with different cutoff threshold.

11. Culture chamber according to claim 10, characterized in that the culture filter membrane M2 rests on the membrane with cutoff threshold of at most 15 Kda.

12. Culture chamber according to one of claims 10 or 11, characterized in that the M2 culture filter membrane is modified by grafting or by cocultures of cells.

13. Culture chamber according to claim 12, characterized in that the modifying grafting of the M2 culture filter membrane relates to the binding of ligands for adhesion molecules of glycoprotein types, of antibodies, of protein molecules.

14. Culture chamber according to claim 12, characterized in that the modification by coculturing of cells is done by formation of a substratum from a first cell type which constitutes the first culture of adherent cells, followed by rinsing of said substrate and the establishment of a second cell type, and optimization of coculture conditions.

15. Culture chamber according to at least one of claims 10 to 14, characterized in that the culture filter membrane M2 forming a biocompatible culture support has a cut-off threshold chosen in the interval ranging from 0.01 μm to 7 .mu.m.

16. Culture chamber according to at least one of claims 1 to 4 and 10 to 15, characterized in that the filter membranes Ml and M3 with different cutoff threshold and the culture filter membrane M2 are supported by mesh supports .

17. Culture chamber according to at least one of claims 1 to 16, characterized in that the internal faces of the end walls of the envelope with axis of symmetry constitute means for distributing dynamic liquid media.

18. Culture chamber according to claim 17, characterized in that the internal faces of the end walls are smooth.

19. Culture chamber according to claim 17, characterized in that the internal faces of the two end walls are provided with distribution ridges of the two dynamic liquid media F1 and F3.

20. Culture chamber according to claim 19, characterized in that the distribution streaks of the end walls are organized in a main network, these streaks being preferably divergent in the direction of propagation of dynamic liquid media.

21. Culture chamber according to at least one of claims 19 to 20, characterized in that the distribution streaks of the end walls are supplemented by a secondary network formed by secondary streaks which are substantially perpendicular to the streaks in the main network.

22. Culture chamber according to either of claims 19 to 21, characterized in that the main and secondary networks constitute a network grid of propagation of dynamic liquid media.

23. Culture chamber according to at least one of claims 19 to 22, characterized in that the streaks of the main network and / or of the secondary network of the end walls are in contact with the membranes with different cut-off thresholds.

24. Culture chamber according to at least one of claims 19 to 22, characterized in that the streaks of the main network have a depth of at most 5 mm, a width of at most 2 mm, and the pitch between two adjacent streaks is at most 2 mm.

25. Culture chamber according to at least one of claims 19 to 23, characterized in that the ridges of the secondary network have a depth of at most 2 mm, a width of at most 2 mm.

26. Culture chamber according to at least one of claims 1 to 25, characterized in that one of the two pairs of inputs and outputs F1 of dynamic liquid media is connected between an end wall and the membrane Ml with cutoff threshold between 0.01 μm and 7 μm and feeds the cell culture medium, in a nutritive medium comprising growth factors by crossing said flat filtering membrane with cutoff threshold between 0.01 and 7 .mu.m.

27. Culture chamber according to claim 26, characterized in that the input and output torque of the dynamic liquid medium F1 operates in a closed circuit according to a loop.

28. Culture chamber according to at least one of claims 1 to 27, characterized in that the input and output torque of the dynamic liquid medium F2, connected between the two filter membranes, assumes the functions of introducing the cells to be cultured and of recovering said cultured cells, of gene transfer and of recovering genetically modified cells, and introduction of a rinsing liquid and elimination of molecules inhibiting the development of cells in culture.

29. Culture chamber according to at least one of claims 1 to 28, characterized in that the other of the two pairs of inputs and outputs of the dynamic liquid medium F3 is connected in open circuit at the level of the other wall end and feeds the cell culture medium in a nutrient medium devoid of growth factors, by crossing the flat filter membrane with cutoff threshold of at most 15 KDa.

30. Culture chamber according to at least one of claims 1 to 29, characterized in that the three pairs of inputs and outputs of dynamic liquid media are placed in three vertical planes passing through the axis of symmetry of the envelope, these planes being offset by an angle of approximately 60 ° between the first inlet and the second inlet, and by an angle of approximately 120 ° between the first and the third inlet of dynamic liquid media, the outlets of said media liquids being in the same angular arrangements.

31. Bioreactor for the extracorporeal culture of animal cells comprising a culture chamber delimited by an envelope with axis of symmetry formed by an external side wall, two end walls and inlets and outlets of dynamic liquid media and comprising ways to circulation of said media in said chamber, characterized in that it comprises:

a) a culture chamber of said cells comprising at least two flat filtering membranes Ml and M3 with different cutoff threshold, perpendicular to the axis of symmetry and that between said membranes with different cutoff threshold is located a means forming a culture support biocompatible promoting the adhesion of cells in a culture state, said chamber being delimited by an envelope with an axis of symmetry comprising two end walls constituting means for distributing dynamic liquid media and three pairs of inputs and outputs of liquid media dynamic F1, F2, F3, intended to supply the culture chamber of the cells and to selectively extract the cultured cells, the waste resulting from their culture and the excess nutrients, two of which are, for each of them, connected between one of the end walls and one of the membranes, the third being connected between the two flat filter membranes your; b) means for circulating the first dynamic liquid medium F1 in a closed loop circuit and an expansion vessel RI containing said medium, these means being connected to said culture chamber; c) means for circulating the second dynamic liquid medium F2 in a closed loop or open loop circuit according to the function assigned to said medium and a reservoir R2 containing said medium, these means being connected to said culture chamber; d) means for circulating the third dynamic liquid medium F3 according to an open loop circuit and a reservoir R3 containing said medium, these means being connected to said culture chamber; e) means of control, regulation, and conditioning of dynamic liquid media, connected to a regulation-command block.

32. Bioreactor according to claim 31, characterized in that the means for circulating said liquid media F1, F2 and F3 are equipped with pumps PI, P2 and P3 respectively, connected to the regulation-command block.

33. Bioreactor according to claims 31 and 32, characterized in that the means for circulating the third dynamic liquid medium F3 are equipped with a valve V3 connected to the regulation-control block.

34. Bioreactor according to claim 31, characterized in that the expansion vessel RI containing the dynamic liquid medium F1 rich in growth factors is provided with a filter having a cut-off threshold of 0.22 μm and with a solenoid valve VI controlled by the regulation-control block.

35. Bioreactor according to at least one of claims 31 to 34, characterized in that the expansion vessel RI is provided with sensors for high level and low level of liquid medium, which serve to trigger the reversal of circulation fluids within said culture chamber.

36. Bioreactor according to claim 31, characterized in that the means for conditioning the dynamic liquid media comprise means for adjusting the oxygen, temperature and pH of the said media.

37. Bioreactor according to claim 36, characterized in that the means for conditioning said media are functional unit modules, dimensioned for a certain mass transfer or thermal energy value.

38. Bioreactor according to claim 37, characterized in that the functional modules are aerators, heat exchangers, pH regulators, or even dialysis modules, ultrafiltration.

39. Bioreactor according to claims 31 to 38, characterized in that it comprises a culture chamber according to at least one of claims 1 to 30.

40. Bioreactor according to at least one of claims 31 to 39, characterized in that a pressure pi prevails in the zone Cl of the culture chamber, situated between the upper end wall and the planar filtering membrane with cut-off threshold between 0.01 μm to 7 μm, from said chamber.

41. Bioreactor according to at least one of claims 31 to 40, characterized in that a pressure p2 prevails in the zone 02 of the culture chamber comprised between the two flat filter membranes with a different cutoff threshold from said chamber.

42. Bioreactor according to at least one of claims 31 to 41, characterized in that a pressure p3 prevails in zone 03 of the culture chamber, situated between the flat filtering membrane with cutoff threshold of at most 15 KDa and the bottom end wall of the envelope ^'of said chamber.

43. Bioreactor according to at least one of claims 31 to 42, characterized in that the nourishing medium rich in growth factors F1 is drained from zone Cl of the culture chamber towards zone C3 of said culture chamber when the pressure pi is higher than the pressure p3 and that the pressure p2 is between pi and p3 in the culture chamber.

44. Bioreactor according to at least one of claims 31 to 42, characterized in that the basic nourishing medium lacking in growth factors F3 is drained from zone 03 of the culture chamber towards zone Cl of said culture chamber when the pressure p3 is greater than the pressure pi and the pressure p2 is between pi and p3 in the culture chamber.

45. Bioreactor according to at least one of claims 31 to 43, characterized in that the opening of the pump PI allows the supply of the culture chamber in feed medium F1 rich in growth factors, the pump P3 being at the stop, and that the backpressure valve V3 is adjusted so that the pressure pi prevailing in the zone C1 of said culture chamber is greater than the pressure p3 prevailing in zone C3 of said chamber, knowing that the pressure p2 prevailing in the zone C2 is between the pressures pi and p3.

46. Bioreactor according to claim 45, characterized in that the regulation-command block programmed according to a particular sequence reverses the direction of flow of flows by stopping the pump PI and by starting the pump P3 as soon as the volume contained in the RI expansion vessel reaches a low level.

47. Bioreactor according to claim 45, characterized in that the valve V3 is closed.

48. Bioreactor according to claims 46 and 47 characterized in that the basic feeding medium F3 devoid of growth factors allows the replenishment of the culture chamber where the pressure p3 in zone C3 of said chamber is greater than the pressure pi prevailing in zone C1, knowing that the pressure p2 prevailing in zone C2 is between the pressures pi and p3.

49. Bioreactor according to at least one of claims 31 to 48, characterized in that the pumps PI and P3 are stopped when the configuration of entry and exit of circulation of the second dynamic liquid medium F2 is fixed in a closed or open circuit depending on the function assigned to it.

50. Bioreactor according to claim 49, characterized in that the dynamic liquid medium F2 is used for the introduction into the culture chamber of the cells to be cultured and for the recovery of the cells after culture.

51. Bioreactor according to claim 49, characterized in that the dynamic liquid medium F2 is used for the introduction of gene transfer vector into the culture chamber containing the cells in culture and for the recovery of the cultured cells, genetically modified.

52. Bioreactor according to claim 49, characterized in that the dynamic liquid medium F2 is used for the introduction of a liquid for rinsing molecules inhibiting the development of cells in culture and for eliminating said molecules.

53. Bioreactor according to claim 50, characterized in that for the inoculation of said medium F2 in zone 02 of the cell culture chamber through a biocompatible septum using a syringe, the solenoid valve V2E1 is open, while the solenoid valves V2E2, V2E3, V2S1, V2S2 and V2S3 are closed.

4. Bioreactor according to claim 50, characterized in that for the recovery of the cells after culture in the culture chamber, the solenoid valve V2S1 is open, the solenoid valves V2E2, V2E3, V2S2, and V2S3 are closed and the pump P2 is switched on on the way to purge the contents of zone 02 in the cell collection pocket.

55. Bioreactor according to claim 52, characterized in that for the elimination of the inhibitory molecules when the second medium F2 contains the elements necessary for rinsing the cell confinement space, during the introduction of said medium the solenoid valve V2E3 is open and the solenoid valves V2E1, V2E2 are closed.

56. Bioreactor according to claim 52, characterized in that this second rinsing medium F2 circulates continuously in open loop and that during the introduction and recovery of said rinsing medium the solenoid valves V2E1, V2E2, V2S1, V2S2 are closed , the solenoid valves V2E3, V2S3 are open.

57. Bioreactor according to claim 51, characterized in that, for the inoculation of the gene transfer vectors contained in said medium F2 circulating in closed circuit in zone 02 of the cell culture chamber, the solenoid valve V2E2 is open, then that the solenoid valves V2E1, V2E3, V2S1, V2S3 are closed.

58. Bioreactor according to at least one of claims 31 to 57, characterized in that the regulation-command block receives all the information relating to the dynamic liquid media F1, F2, F3, by the control and regulation means , and information about the various tanks, pumps, valves and pressures reigning in zones C1, C2, 03 of the culture chamber, manages them and issues the necessary operating orders.