EP0672112A1 - Cell culture or metabolite production device - Google Patents

Cell culture or metabolite production device

Info

Publication number
EP0672112A1
EP0672112A1 EP93913107A EP93913107A EP0672112A1 EP 0672112 A1 EP0672112 A1 EP 0672112A1 EP 93913107 A EP93913107 A EP 93913107A EP 93913107 A EP93913107 A EP 93913107A EP 0672112 A1 EP0672112 A1 EP 0672112A1
Authority
EP
European Patent Office
Prior art keywords
nutritional liquid
culture
permeable
liquid
chamber
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP93913107A
Other languages
German (de)
French (fr)
Inventor
Dominique Cognard
Bernard Dutertre
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bertin Technologies SAS
Original Assignee
Bertin et Cie SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bertin et Cie SA filed Critical Bertin et Cie SA
Publication of EP0672112A1 publication Critical patent/EP0672112A1/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/641—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis

Definitions

  • the invention relates to a cell culture or metabolite production device of the continuously operating type.
  • a cell culture or metabolite production device in which a cell culture chamber is delimited by two microporous membranes between which the cells are confined, and is supplied with nutrients by means of a sheet of tubes with permeable walls in which a flow of nutritional liquid circulates at a pressure higher than that of the liquid medium in the culture chamber.
  • the object of the present invention is in particular to avoid all these drawbacks.
  • It relates to a cell culture or metabolite production device in which the means for supplying cells with nutrients are inexpensive, non-clogable, easily cleanable and sterile. reusable, and easy to assemble.
  • the invention provides a cell culture or metabolite production device, comprising at least one cell culture chamber in a liquid medium, in which two walls are selectively permeable, respectively for introducing fresh nutritional liquid into the culture chamber and the outlet of spent nutritional liquid from this chamber, and means for supplying fresh nutritional liquid under pressure, characterized in that the supply means comprise surfaces which are substantially parallel and adja ⁇ cent, rigid and non-permeable, which delimit between them micro-passages constituting a distributor with uniform flow of nutritional liquid in the culture chamber.
  • the uni ⁇ form distribution of nutritional liquid is no longer made by passing through a microporous wall, but by flow in very small gaps or intervals formed between rigid and non-permeable parallel surfaces, this which reduces the risk of clogging by the nutritional liquid and greatly improves the uniformity of distribution.
  • the micropores that the permeable walls or the porous membranes of the prior art have have an irregular cross-section and a very small surface area, capable of being obstructed very quickly.
  • the micro-passages which the rigid and non-permeable surfaces define between them have a very elongated cross section, of small width and of very great length, so that their obstruction could only occur very gradually and after a very long period of use.
  • the aforementioned surfaces delimiting these micro-passages have a calibrated surface condition and are applied to one another.
  • micro-passages can be machined in the aforementioned surfaces which are applied one on top of the other.
  • a permeable wall according to the invention is then extremely simple, since it suffices to stack or juxtapose surfaces bearing on one another.
  • these surfaces are adjacent faces of compact and non-porous elements which are stacked or juxtaposed, or else nested one inside the other.
  • These elements may be washers stacked axially, or else washers of different diameters, arranged one inside the other in the same plane, or even flat plates of the same dimension, juxtaposed and stacked in a direction perpen --- dicular to their plan.
  • these surfaces are formed by the faces of a ribbon wound with contiguous turns and whose ends are fixed.
  • the ribbon wound in a spiral can be housed in a cavity or an annular bowl of a support element with central hub on which is fixed one end of the ribbon, the other end of the latter being fixed on the peripheral wall. of the aforementioned cavity or cu ⁇ vette. It is then possible to provide two conduits for circulating nutritional liquid opening into the bottom of this bowl, for example one in the vicinity of the peri the latter and the other near the central hub, with a free space for the passage of nutrient liquid made between the bottom of the bowl and the ribbon wound in a spiral.
  • the aforementioned means for supplying fresh nutritional liquid constitute the permeable wall for introduction into the cul ⁇ ture chamber. This avoids using a microporous membrane for cell containment.
  • the permeable wall for the exit of spent nutri ⁇ tional liquid outside the culture chamber can have substantially the same structure as the aforementioned wall for introducing the fresh nutritional liquid.
  • the supply means according to the invention have the extremely important advantage of constituting liquid distributors with uniform flow distributed over a large surface, so that they can be used both for the introduction and the exit of liquid. nutritional than for cell containment, which avoids the use of microporous cell containment membranes provided in the prior art, and at the same time avoids all the disadvantages associated with the use of these membranes.
  • Such a device is characterized in that it comprises at least two cell culture chambers, vertically superimposed and connected to each other, constituted by stacking of alternating annular elements, some of which form the culture chambers and the others of which carry the aforementioned permeable walls for introducing and leaving nutrient liquid and confining cells.
  • FIG. 1 is a schematic view in axial section of a culture device according to the invention
  • - Figure 2 is a schematic sectional view along the line II-II of Figure 1;
  • FIG. 3 is a schematic view illus ⁇ tring the principle of a permeable wall according to the invention
  • FIG. 6 is a schematic view in axial section of a device according to the invention with several culture stages;
  • FIGs 7 and 8 are schematic tick views from above of two annular elements forming part of the device of Figure 6.
  • FIG. 1 and 2 a first embodiment of a device according to the invention, comprising only a culture chamber and which consists essentially of superimposition and sealed assembly of three elements, namely a bottom 10 comprising the means for supplying nutri ⁇ tional liquid, an intermediate element 12 delimiting the culture chamber proper, and an element or upper ca ⁇ pot 14 which are tightly assembled and detachably fixed together, for example by screw- wise.
  • the lower element 10 comprises the means for supplying nutritional liquid, which here consist of two sets 18 of washers 20 axially stacked which extend between two opposite walls of the lower element 10 and which are clamped between two tubular stops 22 mounted in leaktight manner in holes passing through the walls of the lower element 10 to form inlet and outlet ports for nu ⁇ tritional liquid.
  • the washers 20 are kept in axial alignment between three longitudinal rods 24, 120 'from each other and the ends of which are carried by the stops 22.
  • One of these stops comprises a spring 26 exerting pressure on the stack of washers 20.
  • the faces of these washers, which are thus applied one on the other with a determined pressure have a surface condition or a roughness calibrated so as to delimit between them micro-passages of very small width (less at 5 ⁇ m and for example between 1 and 5 ⁇ ) that the nutritional liquid can borrow to flow radially outwards from the axis of the stack of washers 20.
  • the washers 20 can be kept in axial alignment by three longitudinal weld seams, which replace the longitudinal rods 24.
  • the intermediate element 12 delimiting the culture chamber on the periphery, comprises a bundle of small parallel tubes 28 with gas-permeable walls, the ends of which open into two opposite cavities 30 of the annular element 12 communicating with the exterior through through holes 32 of the upper cover 14.
  • These tubes 28 are preferably made of silicone to be completely hydrophobic and to have no adhesion towards the cells.
  • the intermediate element 12 is preferably also made of silicone and is overmolded on the ends of the small tubes 28.
  • the tubes 28 fill substantially all of the internal space of the culture chamber and are separated from each other by short distances of the order of a few millimeters.
  • the upper cover 14 comprises a central orifice 34 for the outlet of spent nutritional liquid and of meta-bolites excreted by the cells. It also comprises, in its periphery, a through orifice 36 which opens into a passage 38 formed in the annular element 12 for bringing the cells into the culture chamber. Another passage 40 formed in the annular element 12 and communicating with a passage of the lower element 10, makes it possible to empty the culture chamber.
  • This device is used in the following way: the passage 40 being closed, a cell culture is brought between the membranes 16 via the orifice 36 and the passage 38, which are then closed.
  • a gas stream essentially containing air circulates in the orifices 32, the cavities 30 and the small tubes 28 with permeable walls as indicated by the arrows, for the supply of oxygen to the cells and the extraction of CO2 produced by cells.
  • a flow of pressurized nutritional liquid circulates in the sets 18 of stacked washers 20, the pressure of this flow being greater than the pressure in the culture chamber, so that a small part of this flow of nutritional liquid flows radially between the stacked washers 20 to pass through the lower mem ⁇ brane 16 and reach the culture chamber, this low flow rate of nutritional liquid being distributed in a uniform manner substantially over the entire surface of the culture chamber.
  • Spent nutritional liquid and metabolites excreted by the cells cross the upper mem ⁇ brane 16 and leave the upper cover by the central orifice 34.
  • the flow of nutritional liquid which circulates in the sets 18 of stacked washers is typically of the order of 10 times the flow which flows between the washers to supply the cells, and its pressure is slightly higher than the pressure in the culture chamber, which is substantially equal to the atmospheric pressure.
  • the flow speed of the nutri ⁇ tional liquid in the culture chamber is very low, for example of the order of 1 to 10 ⁇ m per second.
  • the risks of clogging of the means for supplying nutritional liquid, constituted by the stacks 18 of washers 20, are almost zero.
  • the membranes 16 only have the role of confining the cells, and no longer have a role of determining, distributing and regulating the flow of nutritional liquid.
  • the micro-passages between the ron ⁇ delles 20 can be machined in one face of each washer.
  • One can for example form one or more diametrical or radial V-shaped grooves in one face of each washer over a depth of between 10 and 100 ⁇ for example, as shown in 42 in FIG. 1.
  • FIGS. 3 to 5 represent other embodiments of the means for supplying nu ⁇ tritionnal liquid, which have the further advantage of forming a flat surface ⁇ perpendicular to the direction of flow of the nutritional liquid, which removes mem ⁇ branes 16 for confining cells used in the prior art.
  • These means for supplying nutri ⁇ tional liquid consist essentially of a series of com ⁇ pact elements 44 juxtaposed inside a box 46 comprising a bottom 48 and a peripheral wall 50, the elements ments 44 being spaced from the bottom 48 of the box to leave free a space 52 into which the inlet and outlet conduits for nutritional liquid open, as shown diagrammatically by the arrows 54.
  • the elements 44 juxtaposed inside the box 46 can be flat plates juxtaposed or stacked in a direction perpendicular to their plane. These plates have a surface state or roughness determined to form between them micro-passages of nutritional liquid when they are applied to one another.
  • the peripheral wall 50 of the box 46 is square or rectangular.
  • the elements 44 may be annular rounds with circular outline, of progressively increasing diameters, so as to be able to be nested one inside the other in the same plane.
  • the peripheral wall 50 of the box 46 is circular.
  • the permeable wall is constituted by a strip 56, for example metallic, wound with contiguous turns to form a circular plate with uniform passage rate over its entire surface, one of the extremi tees 58 of this ribbon being fixed on the peripheral wall 50 of the box 46, the other end 60 of the ribbon being fixed on a central hub 62 of the box 46.
  • the ends of the ribbon can be bent at right angles to be introduced into a slot of the central hub 62 and into a slot 64 of the internal face of the wall periphery 50, respectively.
  • the radial dimension of the micro ⁇ passages formed between the turns of the ribbon 56 can be determined by the surface condition or the roughness of the faces of the ribbon, as well as by the tightening or winding of the ribbon on itself.
  • the internal face of the peripheral wall 50 can for this purpose comprise several slots 64 angularly spaced, for the adjustment of this winding.
  • FIG. 6 shows a column reactor which uses the means for supplying nutritional liquid of FIG. 5.
  • the column reactor of FIG. 6 is of the type with several vertically superimposed cell culture chambers and consists essentially by a vertical empi ⁇ lage of alternating annular elements 66 and 68, the elements 66 comprising the permeable walls for introducing and leaving nutritional liquid, the elements 68 forming the actual culture chambers.
  • the elements 66 and 68 have orifices intended to be axially aligned to constitute, when the elements 66 and 68 are stacked and assembled in a sealed manner, vertical conduits 70 and 72 for circulation of fresh nutritional liquid, 74 for the outlet of used nutritional liquid and metabolites, and 76 and 78 for circulating a flow of gas containing oxygen and CO2 ( Figures 7 and 8).
  • annular elements 66 comprise on each of their two large faces, an annular cup 80 with central hub 82, in which a ribbon 84 is wound tightly contiguous turns, as in the embodiment of FIG. 5.
  • the bottom of the bowl 80 has ribs 86 (FIG. 7) making it possible to separate the ribbon 84 from the bottom of the bowl 80.
  • These ribs can be radial as shown, or in a spiral so that the liquid circulates rapidly in a spiral between these ribs and inside the turns of the ribbon 84.
  • Two passages 88 represent link the bottom of the bowl 80 of the upper face of the element 66 to the conduits 70 and 72, respectively.
  • a passage 90 formed in the annular element 66 connects the bottom of the bowl -80 from its underside to the conduit 74.
  • the hubs 82 include an axial passage 92 which passes through the entire annular element 66, to make com ⁇ municate two vertically superimposed culture chambers.
  • the lower annular element 66 only its upper face comprises a bowl receiving a ribbon wound with contiguous turns and communicating with the conduits 70 and 72 for circulation of fresh nutritional liquid.
  • annular bowl 80 in which is disposed a ribbon wound with contiguous turns and which is connected by a passage 90 to the conduit 74 for the outlet of nutritional liquid spent and metabolites.
  • the annular elements 68 forming the culture chambers are of the same type as the annular element 12 of FIGS. 1 and 2 and each comprise a bundle of capillary tubes 94 with walls permeable to gases, made for example of silicone and of which the ends open respectively into the above-mentioned orifices 76 and 78 (FIG. 8).
  • each element 68 can also be made of a cone, and molded onto the ends of the capillary tubes 94.
  • each element 68 mounted clamped between two elements 66 constitutes a cell culture chamber, the upper and lower faces of which are delimited by the ribbons 84 wound in the annular cavities 80 of the lower and upper elements 66.
  • the ru ⁇ ban 84 on the underside of the culture chamber constitutes a permeable liquid supply wall nutritional and cell containment inside the culture chamber.
  • the tape 84 of the upper face of this culture chamber constitutes a permeable wall for the outlet of spent nutritional liquid and of metabolites excreted by the cells, as well as a cell confinement wall inside the culture chamber.
  • the culture chamber which is located on the lower stage of the column reactor has a volume much lower than that of the other cul ⁇ ture chambers of this reactor, to constitute a pre-cul ⁇ ture stage from which we will seed the upper stages of the reactor thanks to the axial passages 92 formed in the annular elements 66.
  • the upper end of the axial passage 92 of the upper element 66 opens out to form a vent hole and comprises a selective closure means 96.
  • the conduit 70 for circulating fresh nutritional liquid opens out to the outside of the upper element 66 to form a vent hole and comprises means 98 for selective closure.
  • conduits 70 and 72 are connected to each other via a flow limitation valve 100 and a circulation pump 102, to form a closed loop for circulation of fresh nutritional liquid, also comprising back-up means 104.
  • Means are also provided for circulating an appropriate gas mixture in the conduits 76 and 78 and the bundles of capillary tubes 94.
  • the lower culture chamber of the reactor having a much lower volume than that of the other culture chambers (from 10 to 1000 times smaller depending on the case) is used for the development of a very sample concentrate of a cell culture, which is introduced into this lower chamber through the axial passage 92 of the lower element 66.
  • the fresh nutritional liquid circulating under the permeable wall 84 of the lower element 66 contains growth factors appropriate, to significantly increase the number of cells.
  • the various culture stages of the column reactor can then be seeded one after the other, by opening the selective sealing means 96 and 98 provided at the upper end of the axial passage 92 of the upper element 66 and at the 'upper end of the duct 70.
  • the constituent elements of the column reactor are either metallic (for example in stainless steel) for the annular elements 66, or in plas ⁇ tic material (for example in silicone) for the annular elements 68. These elements can be tightly assembled by simple compression of annular seals, which may optionally be formed projecting on the opposite faces of the elements 68 in sili ⁇ cone.
  • the cleaning and sterilization of this column reactor is carried out without difficulty, for example by circulation of water vapor in the conduits 70 and 72, as well as in the axial passages 92 of the elements 66.
  • the column reactor is fully removable and therefore easily cleanable.
  • the unclogging of the permeable walls according to the invention can be carried out very simply by thermal shocks, mechanical shocks, vibra ⁇ tions, or by magneto-necking.

Abstract

Cell culture or metabolite production device comprising means for supplying fresh nutritional liquid to a culture chamber. Said means consist, for example, of a stack (18) of annular elements such as disks (20) forming a nutritional liquid circulation tube, the surfaces of said disks constituting with one another micropassages for supplying nutritional liquid to a cellular culture chamber delimited between two microporous membranes (16).

Description

DISPOSITIF DE CULTURE DE CELLULES OO DE PRODUCTION DE DEVICE FOR CULTURING OO CELLS FOR PRODUCING
METABOLITESMETABOLITES
L'invention concerne un dispositif de culture de cellules ou de production de métabolites, du type fonctionnant en continu.The invention relates to a cell culture or metabolite production device of the continuously operating type.
On connaît déjà, par le Brevet FrançaisWe already know, by the French Patent
2 660 323 de la Demanderesse, un dispositif de culture de cellules ou de production de métabolites dans lequel une chambre de culture de cellules est délimitée par deux membranes microporeuses entre lesquelles sont confinées les cellules, et est alimentée en nutriments par l'intermédiaire d'une nappe de tubes à parois perméables dans lesquels circule un débit de liquide nutritionnel à une pression supérieure à celle du milieu liquide dans la chambre de culture.2,660,323 from the Applicant, a cell culture or metabolite production device in which a cell culture chamber is delimited by two microporous membranes between which the cells are confined, and is supplied with nutrients by means of a sheet of tubes with permeable walls in which a flow of nutritional liquid circulates at a pressure higher than that of the liquid medium in the culture chamber.
Sous l'effet de la différence de pression, une partie du débit de nutriments traverse les parois des tubes et circule à faible vitesse dans la chambre de cul¬ ture. Ce système d'alimentation tangentielle (du fait que le débit de nutriments dans les tubes est tangentiel aux parois perméables des tubes d'alimentation de la chambre de culture) présente moins de risques de colmatage des parois perméables par les nutriments que dans la tech¬ nique antérieure où un liquide nutritionnel est dirigé perpendiculairement sur la surface d'une membrane micro¬ poreuse de confinement de cellules.Under the effect of the pressure difference, part of the nutrient flow crosses the walls of the tubes and circulates at low speed in the cul¬ ture chamber. This tangential feed system (because the flow of nutrients in the tubes is tangential to the permeable walls of the supply tubes of the culture chamber) presents less risk of clogging of the permeable walls by the nutrients than in the tech¬ anterior pic where a nutritional liquid is directed perpendicularly to the surface of a micro¬ porous cell confinement membrane.
Ces tubes dont les parois ont une perméabilité contrôlée, ont cependant pour inconvénients d'être très coûteux et sensibles aux chocs thermiques, ce qui pose des problèmes de nettoyage et de stérilisation. De plus, leur montage est délicat et difficile.These tubes, the walls of which have controlled permeability, however have the disadvantages of being very expensive and sensitive to thermal shock, which poses cleaning and sterilization problems. In addition, their assembly is delicate and difficult.
La présente invention a notamment pour but d'éviter tous ces inconvénients.The object of the present invention is in particular to avoid all these drawbacks.
Elle a pour objet un dispositif de culture de cellules ou de production de métabolites dans lequel les moyens d'alimentation des cellules en nutriments sont peu coûteux, non colmatables, facilement nettoyables et sté- rilisables, et de montage simple.It relates to a cell culture or metabolite production device in which the means for supplying cells with nutrients are inexpensive, non-clogable, easily cleanable and sterile. reusable, and easy to assemble.
Elle a également pour objet un dispositif de ce type ne comprenant pas de membranes microporeuses de confinement des cellules. Elle a encore pour objet un dispositif de ce type comprenant plusieurs étages de culture communiquant entre eux, pour constituer ensemble un volume de culture très important et réduire les manipulations.It also relates to a device of this type not comprising microporous membranes for confining cells. It also relates to a device of this type comprising several culture stages communicating with each other, to together constitute a very large culture volume and reduce handling.
L'invention propose un dispositif de culture de cellules ou de production de métabolites, comprenant au moins une chambre de culture cellulaire en milieu li¬ quide, dans laquelle sont prévues deux parois sélective¬ ment perméables, respectivement d'introduction de liquide nutritionnel frais dans la chambre de culture et de sor- tie de liquide nutritionnel usé de cette chambre, et des moyens d'alimentation en liquide nutritionnel frais sous pression, caractérisé en ce que les moyens d'alimentation comportent des surfaces sensiblement parallèles et adja¬ centes, rigides et non perméables, qui délimitent entre elles des micro-passages constituant un distributeur à débit uniforme de liquide nutritionnel dans la chambre de culture.The invention provides a cell culture or metabolite production device, comprising at least one cell culture chamber in a liquid medium, in which two walls are selectively permeable, respectively for introducing fresh nutritional liquid into the culture chamber and the outlet of spent nutritional liquid from this chamber, and means for supplying fresh nutritional liquid under pressure, characterized in that the supply means comprise surfaces which are substantially parallel and adja¬ cent, rigid and non-permeable, which delimit between them micro-passages constituting a distributor with uniform flow of nutritional liquid in the culture chamber.
Ainsi, selon l'invention, la distribution uni¬ forme de liquide nutritionnel ne se fait plus par traver- sée d'une paroi microporeuse, mais par écoulement dans des jeux ou intervalles très faibles formés entre des surfaces parallèles rigides et non perméables, ce qui ré¬ duit les risques de colmatage par le liquide nutritionnel et améliore largement l'uniformité de la distribution. En effet, les micropores que présentent les parois perméables ou les membranes poreuses de la tech¬ nique antérieure, ont une section transversale irrégu¬ lière et de surface très faible, susceptible d'être obs¬ truée très rapidement. Au contraire, dans la présente in- vention, les micro-passages que définissent entre elles les surfaces rigides et non perméables ont une section transversale très allongée, de largeur faible et de très grande longueur, de sorte que leur obstruction ne pour¬ rait se produire que de façon très progressive et après une très longue durée d'utilisation.Thus, according to the invention, the uni¬ form distribution of nutritional liquid is no longer made by passing through a microporous wall, but by flow in very small gaps or intervals formed between rigid and non-permeable parallel surfaces, this which reduces the risk of clogging by the nutritional liquid and greatly improves the uniformity of distribution. Indeed, the micropores that the permeable walls or the porous membranes of the prior art have, have an irregular cross-section and a very small surface area, capable of being obstructed very quickly. On the contrary, in the present invention, the micro-passages which the rigid and non-permeable surfaces define between them have a very elongated cross section, of small width and of very great length, so that their obstruction could only occur very gradually and after a very long period of use.
Selon un. mode de réalisation de l'invention, les surfaces précitées délimitant ces micro-passages ont un état de surface calibré et sont appliquées les unes sur les autres.According to one. embodiment of the invention, the aforementioned surfaces delimiting these micro-passages have a calibrated surface condition and are applied to one another.
En variante, les micro-passages peuvent être usinés dans les surfaces précitées qui sont appliquées les unes sur les autres.Alternatively, the micro-passages can be machined in the aforementioned surfaces which are applied one on top of the other.
La constitution d'une paroi perméable selon l'invention est alors extrêmement simple, puisqu'il suf¬ fit d'empiler ou de juxtaposer des surfaces en appui les unes sur les autres. Dans une forme de réalisation de l'invention, ces surfaces sont des faces adjacentes d'éléments com¬ pacts et non poreux, qui sont empilés ou juxtaposés, ou bien imbriqués les uns dans les autres.The constitution of a permeable wall according to the invention is then extremely simple, since it suffices to stack or juxtapose surfaces bearing on one another. In one embodiment of the invention, these surfaces are adjacent faces of compact and non-porous elements which are stacked or juxtaposed, or else nested one inside the other.
Ces éléments peuvent être des rondelles empi- lées axialement, ou bien des rondelles de diamètres dif¬ férents, disposées les unes à l'intérieur des autres dans un même plan, ou encore des plaques planes de même dimen¬ sion, juxtaposées et empilées dans une direction perpen--- diculaire à leur plan. Dans une autre forme de réalisation de l'invention, ces surfaces sont formées par les faces d'un ruban enroulé à spires jointives et dont les extrémités sont fixes.These elements may be washers stacked axially, or else washers of different diameters, arranged one inside the other in the same plane, or even flat plates of the same dimension, juxtaposed and stacked in a direction perpen --- dicular to their plan. In another embodiment of the invention, these surfaces are formed by the faces of a ribbon wound with contiguous turns and whose ends are fixed.
Dans ce cas, le ruban enroulé en spirale peut être logé dans une cavité ou une cuvette annulaire d'un élément de support à moyeu central sur lequel est fixée une extrémité du ruban, l'autre extrémité de ce dernier étant fixée sur la paroi périphérique de la cavité ou cu¬ vette précitée. On peut alors prévoir deux conduits de circu¬ lation de liquide nutritionnel débouchant dans le fond de cette cuvette, par exemple l'un au voisinage de la péri- phérie de celle-ci et l'autre au voisinage du moyeu cen¬ tral, avec un espace libre de passage de liquide nutri¬ tionnel ménagé entre le fond de la cuvette et le ruban enroulé en spirale. - Avantageusement, les moyens précités d'alimentation en liquide nutritionnel frais constituent la paroi perméable d'introduction dans la chambre de cul¬ ture. Cela évite d'utiliser une membrane microporeuse pour le confinement des cellules. La paroi perméable de sortie de liquide nutri¬ tionnel usé hors de la chambre de culture peut avoir sen¬ siblement la même structure que la paroi précitée d'introduction du liquide nutritionnel frais.In this case, the ribbon wound in a spiral can be housed in a cavity or an annular bowl of a support element with central hub on which is fixed one end of the ribbon, the other end of the latter being fixed on the peripheral wall. of the aforementioned cavity or cu¬ vette. It is then possible to provide two conduits for circulating nutritional liquid opening into the bottom of this bowl, for example one in the vicinity of the peri the latter and the other near the central hub, with a free space for the passage of nutrient liquid made between the bottom of the bowl and the ribbon wound in a spiral. Advantageously, the aforementioned means for supplying fresh nutritional liquid constitute the permeable wall for introduction into the cul¬ ture chamber. This avoids using a microporous membrane for cell containment. The permeable wall for the exit of spent nutri¬ tional liquid outside the culture chamber can have substantially the same structure as the aforementioned wall for introducing the fresh nutritional liquid.
Les moyens d'alimentation selon l'invention présentent l'avantage extrêmement important de constituer des distributeurs de liquide à débit uniforme réparti sur une surface importante, de telle sorte qu'on peut les utiliser aussi bien pour l'introduction et la sortie de liquide nutritionnel que pour le confinement des cel- Iules, ce qui évite d'utiliser les membranes micropo- reuses de confinement de cellules prévues dans la tech¬ nique antérieure, et évite du même coup tous les inconvé¬ nients liés à l'utilisation de ces membranes.The supply means according to the invention have the extremely important advantage of constituting liquid distributors with uniform flow distributed over a large surface, so that they can be used both for the introduction and the exit of liquid. nutritional than for cell containment, which avoids the use of microporous cell containment membranes provided in the prior art, and at the same time avoids all the disadvantages associated with the use of these membranes.
Il devient alors possible de réaliser simple- ment un dispositif de culture à plusieurs étages dont le fonctionnement est particulièrement fiable et qui pré¬ sente de grands avantages au niveau du nettoyage et de la stérilisation.It then becomes possible to simply produce a multi-stage culture device whose operation is particularly reliable and which has great advantages in terms of cleaning and sterilization.
Un tel dispositif selon l'invention est carac- térisé en ce qu'il comprend au moins deux chambres de culture cellulaire, verticalement superposées et reliées l'une à l'autre, constituées par empilage d'éléments an¬ nulaires alternés dont les uns forment les chambres de culture et dont les autres portent les parois perméables précitées d'introduction et de sortie de liquide nutri¬ tionnel et de confinement de cellules.Such a device according to the invention is characterized in that it comprises at least two cell culture chambers, vertically superimposed and connected to each other, constituted by stacking of alternating annular elements, some of which form the culture chambers and the others of which carry the aforementioned permeable walls for introducing and leaving nutrient liquid and confining cells.
On obtient ainsi un dispositif à volume de culture évolutif, qui permet notamment de commencer une culture dans une chambre de culture inférieure, de volume relativement très faible, puis d'augmenter le volume de culture au fur et à- mesure de la croissance et du déve- loppement des cellules. On évite alors toutes les manipu¬ lations de transfert de culture, qui étaient nécessaires dans la technique antérieure.This gives a device with a volume of evolutionary culture, which in particular makes it possible to start a culture in a lower culture chamber, of relatively very small volume, then to increase the culture volume as the cells grow and develop. All the culture transfer manipulations which were necessary in the prior art are then avoided.
L'invention sera mieux comprise, et d'autres caractéristiques, détails et avantages de celle-ci appa- raîtront plus clairement à la lecture de la description qui suit, faite à titre d'exemple en référence aux des¬ sins annexés, dans lesquels :The invention will be better understood, and other characteristics, details and advantages thereof will appear more clearly on reading the description which follows, given by way of example with reference to the accompanying drawings, in which :
- la figure 1 est une vue schématique en coupe axiale d'un dispositif de culture selon l'invention ; - la figure 2 est une vue schématique en coupe selon la ligne II-II de la figure 1 ;- Figure 1 is a schematic view in axial section of a culture device according to the invention; - Figure 2 is a schematic sectional view along the line II-II of Figure 1;
- la figure 3 est une vue schématique illus¬ trant le principe d'une paroi perméable selon l'invention- Figure 3 is a schematic view illus¬ tring the principle of a permeable wall according to the invention
- les figures 4 et 5 représentent schématique- ment deux modes de réalisation de cette paroi ;- Figures 4 and 5 schematically represent two embodiments of this wall;
- la figure 6 est une vue schématique en coupe axiale d'un dispositif selon l'invention à plusieurs étages de culture ; - les figures 7 et 8 sont des vues schéma¬ tiques de dessus de deux éléments annulaires faisant par¬ tie du dispositif de la figure 6.- Figure 6 is a schematic view in axial section of a device according to the invention with several culture stages; - Figures 7 and 8 are schematic tick views from above of two annular elements forming part of the device of Figure 6.
On a représenté dans les figures 1 et 2 une première forme de réalisation d'un dispositif selon l'invention, ne comprenant qu'une chambre de culture et qui est constitué essentiellement par superposition et assemblage étanche de trois éléments, à savoir un fond 10 comprenant les moyens d'alimentation en liquide nutri¬ tionnel, un élément intermédiaire 12 délimitant la chambre de culture proprement dite, et un élément ou ca¬ pot supérieur 14 qui sont assemblés de façon étanche et fixés entre eux de façon démontable, par exemple par vis- sage.There is shown in Figures 1 and 2 a first embodiment of a device according to the invention, comprising only a culture chamber and which consists essentially of superimposition and sealed assembly of three elements, namely a bottom 10 comprising the means for supplying nutri¬ tional liquid, an intermediate element 12 delimiting the culture chamber proper, and an element or upper ca¬ pot 14 which are tightly assembled and detachably fixed together, for example by screw- wise.
Deux membranes microporeuses 16 de confinement de cellules sont interposées entre l'élément inférieur 10, l'élément intermédiaire 12, et le capot supérieur 14. L'élément inférieur 10 comprend les moyens d'alimentation en liquide nutritionnel, qui sont ici constitués par deux ensembles 18 de rondelles 20 empilées axialement qui s'étendent entre deux parois opposées de l'élément inférieur 10 et qui sont serrées entre deux bu- tées tubulaires 22 montées à étanchéité dans des orifices traversants des parois de l'élément inférieur 10 pour former des orifices d'entrée et de sortie de liquide nu¬ tritionnel.Two microporous membranes 16 for confining cells are interposed between the lower element 10, the intermediate element 12, and the upper cover 14. The lower element 10 comprises the means for supplying nutritional liquid, which here consist of two sets 18 of washers 20 axially stacked which extend between two opposite walls of the lower element 10 and which are clamped between two tubular stops 22 mounted in leaktight manner in holes passing through the walls of the lower element 10 to form inlet and outlet ports for nu¬ tritional liquid.
Les rondelles 20 sont maintenues en alignement axial entre trois tiges longitudinales 24, à 120' les unes des autres et dont les extrémités sont portées par les butées 22. L'une de ces butées comprend un ressort 26 exerçant une pression sur l'empilage de rondelles 20. Les faces de ces rondelles, qui sont ainsi appliquées les unes sur les autres avec une pression déterminée, ont un état de surface ou une rugosité calibrée de façon à déli¬ miter entre elles des micro-passages de largeur très faible (inférieure à 5 μm et par exemple comprise entre 1 et 5 μ ) que le liquide nutritionnel peut emprunter pour s'écouler radialement vers l'extérieur depuis l'axe de l'empilage de rondelles 20.The washers 20 are kept in axial alignment between three longitudinal rods 24, 120 'from each other and the ends of which are carried by the stops 22. One of these stops comprises a spring 26 exerting pressure on the stack of washers 20. The faces of these washers, which are thus applied one on the other with a determined pressure, have a surface condition or a roughness calibrated so as to delimit between them micro-passages of very small width (less at 5 μm and for example between 1 and 5 μ) that the nutritional liquid can borrow to flow radially outwards from the axis of the stack of washers 20.
En variante, les rondelles 20 peuvent être maintenues en alignement axial par trois cordons longitu¬ dinaux de soudure, qui remplacent les tiges longitudi- nales 24.As a variant, the washers 20 can be kept in axial alignment by three longitudinal weld seams, which replace the longitudinal rods 24.
L'élément intermédiaire 12, délimitant péri- phériquement la chambre de culture, comprend un faisceau de petits tubes parallèles 28 à parois perméables aux gaz, dont les extrémités débouchent dans deux cavités op- posées 30 de l'élément annulaire 12 communiquant avec l'extérieur par des orifices traversants 32 du capot su¬ périeur 14. Ces tubes 28 sont de préférence en silicone pour être totalement hydrophobes et ne présenter aucune adhérence vis-à-vis des cellules. L'élément intermédiaire 12 est de préférence réalisé également en silicone et est surmoulé sur les extrémités des petits tubes 28. Les tubes 28 garnissent sensiblement tout l'espace interne de la chambre de culture et sont séparés les uns des autres par des distances faibles de l'ordre de quelques millimètres .The intermediate element 12, delimiting the culture chamber on the periphery, comprises a bundle of small parallel tubes 28 with gas-permeable walls, the ends of which open into two opposite cavities 30 of the annular element 12 communicating with the exterior through through holes 32 of the upper cover 14. These tubes 28 are preferably made of silicone to be completely hydrophobic and to have no adhesion towards the cells. The intermediate element 12 is preferably also made of silicone and is overmolded on the ends of the small tubes 28. The tubes 28 fill substantially all of the internal space of the culture chamber and are separated from each other by short distances of the order of a few millimeters.
Le capot supérieur 14 comporte un orifice cen- tral 34 de sortie de liquide nutritionnel usé et de méta¬ bolites excrétés par les cellules. Il comprend également, dans sa périphérie, un orifice traversant 36 qui débouche dans un passage 38 formé dans l'élément annulaire 12 pour l'amenée des cellules dans la chambre de culture. Un autre passage 40 formé dans l'élément annulaire 12 et communiquant avec un passage de l'élément inférieur 10, permet de vider la chambre de culture.The upper cover 14 comprises a central orifice 34 for the outlet of spent nutritional liquid and of meta-bolites excreted by the cells. It also comprises, in its periphery, a through orifice 36 which opens into a passage 38 formed in the annular element 12 for bringing the cells into the culture chamber. Another passage 40 formed in the annular element 12 and communicating with a passage of the lower element 10, makes it possible to empty the culture chamber.
Ce dispositif est utilisé de la façon sui¬ vante : - le passage 40 étant obturé, une culture de cellules est amenée entre les membranes 16 par l'intermédiaire de l'orifice 36 et du passage 38, qui sont ensuite obturés . Un courant de gaz contenant essen¬ tiellement de l'air circule dans les orifices 32, les ca- vités 30 et les petits tubes 28 à parois perméables comme indiqué par les flèches, pour l'amenée d'oxygène aux cel¬ lules et l'extraction du CO2 produit par les cellules. Un débit de liquide nutritionnel sous pression circule dans les ensembles 18 de rondelles empilées 20, la pression de cet écoulement étant supérieure à la pression dans la chambre de culture, de sorte qu'une faible partie de ce débit de liquide nutritionnel s'écoule radialement entre les rondelles empilées 20 pour passer à travers la mem¬ brane inférieure 16 et gagner la chambre de culture, ce faible débit de liquide nutritionnel étant réparti de fa¬ çon uniforme sensiblement sur toute la surface de la chambre de culture. Le liquide nutritionnel usé et les métabolites excrétés par les cellules traversent la mem¬ brane supérieure 16 et sortent du capot supérieur par l'orifice central 34.This device is used in the following way: the passage 40 being closed, a cell culture is brought between the membranes 16 via the orifice 36 and the passage 38, which are then closed. A gas stream essentially containing air circulates in the orifices 32, the cavities 30 and the small tubes 28 with permeable walls as indicated by the arrows, for the supply of oxygen to the cells and the extraction of CO2 produced by cells. A flow of pressurized nutritional liquid circulates in the sets 18 of stacked washers 20, the pressure of this flow being greater than the pressure in the culture chamber, so that a small part of this flow of nutritional liquid flows radially between the stacked washers 20 to pass through the lower mem¬ brane 16 and reach the culture chamber, this low flow rate of nutritional liquid being distributed in a uniform manner substantially over the entire surface of the culture chamber. Spent nutritional liquid and metabolites excreted by the cells cross the upper mem¬ brane 16 and leave the upper cover by the central orifice 34.
Le débit -de liquide nutritionnel qui circule dans les ensembles 18 de rondelles empilées est typique¬ ment de l'ordre de 10 fois le débit qui s'écoule entre les rondelles pour alimenter les cellules, et sa pression est légèrement supérieure à la pression dans la chambre de culture, qui est sensiblement égale à la pression at- mospherique. La vitesse d'écoulement du liquide nutri¬ tionnel dans la chambre de culture est très faible, par exemple de l'ordre de 1 à 10 μm par seconde.The flow of nutritional liquid which circulates in the sets 18 of stacked washers is typically of the order of 10 times the flow which flows between the washers to supply the cells, and its pressure is slightly higher than the pressure in the culture chamber, which is substantially equal to the atmospheric pressure. The flow speed of the nutri¬ tional liquid in the culture chamber is very low, for example of the order of 1 to 10 μm per second.
Dans ce dispositif, les risques de colmatage des moyens d'alimentation en liquide nutritionnel, constitués par les empilages 18 de rondelles 20, sont quasi nuls. Les membranes 16 ont uniquement un rôle de confinement des cellules, et n'ont plus un rôle de déter¬ mination, de distribution et de régulation du débit de liquide nutritionnel. En variante, les micro-passages entre les ron¬ delles 20 peuvent être usinés dans une face de chaque rondelle. On peut par exemple former une ou plusieurs rainures diamétrales ou radiales en V dans une face de chaque rondelle sur une profondeur comprise entre 10 et 100 μ par exemple, comme représenté en 42 en figure 1.In this device, the risks of clogging of the means for supplying nutritional liquid, constituted by the stacks 18 of washers 20, are almost zero. The membranes 16 only have the role of confining the cells, and no longer have a role of determining, distributing and regulating the flow of nutritional liquid. Alternatively, the micro-passages between the ron¬ delles 20 can be machined in one face of each washer. One can for example form one or more diametrical or radial V-shaped grooves in one face of each washer over a depth of between 10 and 100 μ for example, as shown in 42 in FIG. 1.
Les figures 3 à 5 représentent d'autres modes de réalisation des moyens d'alimentation en liquide nu¬ tritionnel, qui ont de plus l'avantage de former une sur¬ face plane perpendiculaire à la direction d'écoulement du liquide nutritionnel, ce qui permet de supprimer les mem¬ branes 16 de confinement des cellules utilisées dans la technique antérieure.FIGS. 3 to 5 represent other embodiments of the means for supplying nu¬ tritionnal liquid, which have the further advantage of forming a flat surface¬ perpendicular to the direction of flow of the nutritional liquid, which removes mem¬ branes 16 for confining cells used in the prior art.
Ces moyens d'alimentation en liquide nutri¬ tionnel, représentés en coupe axiale en figure 3, sont constitués essentiellement par une série d'éléments com¬ pacts 44 juxtaposés à l'intérieur d'une boîte 46 compor¬ tant un fond 48 et une paroi périphérique 50, les élé- ments 44 étant espacés du fond 48 de la boîte pour lais¬ ser libre un espace 52 dans lequel débouchent des conduits d'entrée et de sortie de liquide nutritionnel, comme indiqué schématiquement par les flèches 54. Les éléments 44 juxtaposés à l'intérieur de la boîte 46 peuvent être des plaques planes juxtaposées ou empilées dans une direction perpendiculaire à leur plan. Ces plaques ont un état de surface ou une rugosité déter¬ minée pour former entre elles des micro-passages de li- quide nutritionnel lorsqu'elles sont appliquées les unes sur les autres. Dans ce cas, la paroi périphérique 50 de la boîte 46 est de forme carrée ou rectangulaire.These means for supplying nutri¬ tional liquid, represented in axial section in FIG. 3, consist essentially of a series of com¬ pact elements 44 juxtaposed inside a box 46 comprising a bottom 48 and a peripheral wall 50, the elements ments 44 being spaced from the bottom 48 of the box to leave free a space 52 into which the inlet and outlet conduits for nutritional liquid open, as shown diagrammatically by the arrows 54. The elements 44 juxtaposed inside the box 46 can be flat plates juxtaposed or stacked in a direction perpendicular to their plane. These plates have a surface state or roughness determined to form between them micro-passages of nutritional liquid when they are applied to one another. In this case, the peripheral wall 50 of the box 46 is square or rectangular.
En variante, et comme représenté schématique¬ ment en figure 4, les éléments 44 peuvent être des ron- délies annulaires à contour circulaire, de diamètres pro¬ gressivement croissants, de façon à pouvoir être imbri¬ quées les unes à 1'intérieur des autres dans un même plan. Dans ce cas, la paroi périphérique 50 de la boîte 46 est de forme circulaire. Selon une autre variante, représentée schéma¬ tiquement en figure 5, la paroi perméable est constituée par un ruban 56 par exemple métallique enroulé à spires jointives pour former une plaque circulaire à taux de passage uniforme sur toute sa surface, l'une des extrémi- tés 58 de ce ruban étant fixée sur la paroi périphérique 50 de la boîte 46, l'autre extrémité 60 du ruban étant fixée sur un moyeu central 62 de la boîte 46. Les extré¬ mités du ruban peuvent être coudées à angle droit pour être introduites dans une fente du moyeu central 62 et dans une fente 64 de la face interne de la paroi périphé¬ rique 50, respectivement.As a variant, and as shown schematically in FIG. 4, the elements 44 may be annular rounds with circular outline, of progressively increasing diameters, so as to be able to be nested one inside the other in the same plane. In this case, the peripheral wall 50 of the box 46 is circular. According to another variant, shown schematically in FIG. 5, the permeable wall is constituted by a strip 56, for example metallic, wound with contiguous turns to form a circular plate with uniform passage rate over its entire surface, one of the extremi tees 58 of this ribbon being fixed on the peripheral wall 50 of the box 46, the other end 60 of the ribbon being fixed on a central hub 62 of the box 46. The ends of the ribbon can be bent at right angles to be introduced into a slot of the central hub 62 and into a slot 64 of the internal face of the wall periphery 50, respectively.
Dans ce cas, la dimension radiale des micro¬ passages formés entre les spires du ruban 56 peut être déterminée par l'état de surface ou la rugosité des faces du ruban, ainsi que par le serrage ou l'enroulement du ruban sur lui-même. La face interne de la paroi périphé¬ rique 50 peut comprendre à cet effet plusieurs fentes 64 angulairement espacées, pour le réglage de cet enroule¬ ment.In this case, the radial dimension of the micro¬ passages formed between the turns of the ribbon 56 can be determined by the surface condition or the roughness of the faces of the ribbon, as well as by the tightening or winding of the ribbon on itself. . The internal face of the peripheral wall 50 can for this purpose comprise several slots 64 angularly spaced, for the adjustment of this winding.
On peut aussi usiner ou former à intervalles réguliers des rainur-es transversales dans une face de ce ruban, qui sera alors enroulé sur lui-même de façon ser¬ rée.It is also possible to machine or form transverse grooves at regular intervals in one face of this strip, which will then be wound on itself in a serely manner.
On a représenté, en figure 6, un réacteur co¬ lonne qui utilise les moyens d'alimentation en liquide nutritionnel de la figure 5. Le réacteur colonne de la figure 6 est du type à plusieurs chambres de culture cellulaire verticalement superposées et est constitué essentiellement par un empi¬ lage vertical d'éléments annulaires alternés 66 et 68, les éléments 66 comportant les parois perméables d'introduction et de sortie de liquide nutritionnel, les éléments 68 formant les chambres de culture proprement dites.FIG. 6 shows a column reactor which uses the means for supplying nutritional liquid of FIG. 5. The column reactor of FIG. 6 is of the type with several vertically superimposed cell culture chambers and consists essentially by a vertical empi¬ lage of alternating annular elements 66 and 68, the elements 66 comprising the permeable walls for introducing and leaving nutritional liquid, the elements 68 forming the actual culture chambers.
Les éléments 66 et 68 comportent des orifices destinés à être axialement alignés pour constituer, quand les éléments 66 et 68 sont empilés et assemblés de façon étanche, des conduits verticaux 70 et 72 de circulation de liquide nutritionnel frais, 74 de sortie de liquide nutritionnel usé et de métabolites, et 76 et 78 de circu¬ lation d'un débit de gaz contenant de l'oxygène et du CO2 ( figures 7 et 8) .The elements 66 and 68 have orifices intended to be axially aligned to constitute, when the elements 66 and 68 are stacked and assembled in a sealed manner, vertical conduits 70 and 72 for circulation of fresh nutritional liquid, 74 for the outlet of used nutritional liquid and metabolites, and 76 and 78 for circulating a flow of gas containing oxygen and CO2 (Figures 7 and 8).
Les éléments annulaires 66, à l'exception de l'élément inférieur et de l'élément supérieur, comportent sur chacune de leurs deux grandes faces, une cuvette an¬ nulaire 80 à moyeu central 82, dans laquelle un ruban 84 est enroulé serré à spires jointives, comme dans le mode de réalisation de la figure 5.The annular elements 66, with the exception of the lower element and the upper element, comprise on each of their two large faces, an annular cup 80 with central hub 82, in which a ribbon 84 is wound tightly contiguous turns, as in the embodiment of FIG. 5.
Le fond de la cuvette 80 comporte des nervures 86 (figure 7) permettant d'écarter le ruban 84 du fond de la cuvette 80. Ces nervures peuvent être radiales comme représenté, ou en spirale de façon à ce que le liquide circule rapidement en spirale entre ces nervures et à l'intérieur des spires du ruban 84. Deux passages 88 re- lient le fond de la cuvette 80 de la face supérieure de l'élément 66 aux conduits 70 et 72, respectivement. Un passage 90 formé dans l'élément annulaire 66 relie le fond de la cuvette -80 de sa face inférieure au conduit 74. Enfin, les moyeux 82 comportent un passage axial 92 qui traverse tout l'élément annulaire 66, pour faire com¬ muniquer deux chambres de culture verticalement superpo¬ sées.The bottom of the bowl 80 has ribs 86 (FIG. 7) making it possible to separate the ribbon 84 from the bottom of the bowl 80. These ribs can be radial as shown, or in a spiral so that the liquid circulates rapidly in a spiral between these ribs and inside the turns of the ribbon 84. Two passages 88 represent link the bottom of the bowl 80 of the upper face of the element 66 to the conduits 70 and 72, respectively. A passage 90 formed in the annular element 66 connects the bottom of the bowl -80 from its underside to the conduit 74. Finally, the hubs 82 include an axial passage 92 which passes through the entire annular element 66, to make com¬ municate two vertically superimposed culture chambers.
En ce qui concerne l'élément annulaire 66 in- férieur, seule sa face supérieure comporte une cuvette recevant un ruban enroulé à spires jointives et communi¬ quant avec les conduits 70 et 72 de circulation de li¬ quide nutritionnel frais.As regards the lower annular element 66, only its upper face comprises a bowl receiving a ribbon wound with contiguous turns and communicating with the conduits 70 and 72 for circulation of fresh nutritional liquid.
Quant à l'élément supérieur 66 du réacteur co- lonne, seule sa face inférieure comporte une cuvette an¬ nulaire 80 dans laquelle est disposé un ruban enroulé à spires jointives et qui est reliée par un passage 90 au conduit 74 de sortie de liquide nutritionnel usé et de métabolites. Les éléments annulaires 68 formant les chambres de culture sont du même type que 1 'élément annu¬ laire 12 des figures 1 et 2 et comportent chacun un fais¬ ceau de tubes capillaires 94 à parois perméables aux gaz, réalisés par exemple en silicone et dont les extrémités débouchent respectivement dans les orifices 76 et 78 pré¬ cités (figure 8) .As for the upper element 66 of the column reactor, only its lower face comprises an annular bowl 80 in which is disposed a ribbon wound with contiguous turns and which is connected by a passage 90 to the conduit 74 for the outlet of nutritional liquid spent and metabolites. The annular elements 68 forming the culture chambers are of the same type as the annular element 12 of FIGS. 1 and 2 and each comprise a bundle of capillary tubes 94 with walls permeable to gases, made for example of silicone and of which the ends open respectively into the above-mentioned orifices 76 and 78 (FIG. 8).
Comme dans le mode de réalisation de la figure 1, chaque élément 68 peut être également réalisé en sili¬ cone, et surmoulé sur les extrémités des tubes capil- laires 94.As in the embodiment of FIG. 1, each element 68 can also be made of a cone, and molded onto the ends of the capillary tubes 94.
Ainsi, chaque élément 68 monté serré entre deux éléments 66 constitue une chambre de culture cellu¬ laire dont les faces supérieure et inférieure sont déli¬ mitées par les rubans 84 enroulés dans les cavités annu- laires 80 des éléments 66 inférieur et supérieur. Le ru¬ ban 84 de la face inférieure de la chambre de culture constitue une paroi perméable d'alimentation en liquide nutritionnel frais et de confinement des cellules à l'intérieur de la chambre de culture. Le ruban 84 de la face supérieure de cette chambre de culture constitue une paroi perméable de sortie de liquide nutritionnel usé et de métabolites excrétés par les cellules, ainsi qu'une paroi de confinement des cellules à 1'intérieur de la chambre de culture.Thus, each element 68 mounted clamped between two elements 66 constitutes a cell culture chamber, the upper and lower faces of which are delimited by the ribbons 84 wound in the annular cavities 80 of the lower and upper elements 66. The ru¬ ban 84 on the underside of the culture chamber constitutes a permeable liquid supply wall nutritional and cell containment inside the culture chamber. The tape 84 of the upper face of this culture chamber constitutes a permeable wall for the outlet of spent nutritional liquid and of metabolites excreted by the cells, as well as a cell confinement wall inside the culture chamber.
Avantageusement, la chambre de culture qui se trouve à l'étage inférieur du réacteur colonne a un vo- lume très inférieur à celui des autres chambres de cul¬ ture de ce réacteur, pour constituer un étage de pré-cul¬ ture à partir duquel on va ensemencer les étages supé¬ rieurs du réacteur grâce aux passages axiaux 92 formés dans les éléments annulaires 66. L'extrémité supérieure du passage axial 92 de l'élément supérieur 66 débouche à l'extérieur pour former un trou d'évent et comporte un moyen 96 d'obturation sé¬ lective. De même, le conduit 70 de circulation de liquide nutritionnel frais débouche à l'extérieur de l'élément supérieur 66 pour former un trou d'évent et comporte un moyen 98 d'obturation sélective.Advantageously, the culture chamber which is located on the lower stage of the column reactor has a volume much lower than that of the other cul¬ ture chambers of this reactor, to constitute a pre-cul¬ ture stage from which we will seed the upper stages of the reactor thanks to the axial passages 92 formed in the annular elements 66. The upper end of the axial passage 92 of the upper element 66 opens out to form a vent hole and comprises a selective closure means 96. Likewise, the conduit 70 for circulating fresh nutritional liquid opens out to the outside of the upper element 66 to form a vent hole and comprises means 98 for selective closure.
Les extrémités inférieures des conduits 70 et 72 sont reliées entre elles par l'intermédiaire d'une vanne 100 de limitation de débit et d'une pompe de circu- lation 102, pour former une boucle fermée de circulation de liquide nutritionnel frais, comprenant également des moyens d'appoint 104.The lower ends of the conduits 70 and 72 are connected to each other via a flow limitation valve 100 and a circulation pump 102, to form a closed loop for circulation of fresh nutritional liquid, also comprising back-up means 104.
Des moyens, non représentés, sont également prévus pour faire circuler un mélange gazeux approprié dans les conduits 76 et 78 et les faisceaux de tubes ca¬ pillaires 94.Means, not shown, are also provided for circulating an appropriate gas mixture in the conduits 76 and 78 and the bundles of capillary tubes 94.
Le réacteur colonne qui vient d'être décrit est utilisé de la façon suivante :The column reactor which has just been described is used as follows:
La chambre inférieure de culture du réacteur, ayant un volume beaucoup plus faible que celui des autres chambres de culture (de 10 à 1000 fois plus faible selon les cas) sert au développement d'un échantillon très concentré d'une culture de cellules, qui est introduit dans cette chambre inférieure par le passage axial 92 de l'élément inférieur 66. Le liquide nutritionnel frais circulant sous la paroi perméable 84 de 1 'élément infé- rieur 66 contient des facteurs de croissance appropriés, pour augmenter de façon importante le nombre de cellules. Les divers étages de culture du réacteur colonne peuvent ensuite être ensemencés l'un après l'autre, par ouverture des moyens d'obturation sélective 96 et 98 prévus à l'extrémité supérieure du passage axial 92 de l'élément supérieur 66 et à l'extrémité supérieure du conduit 70.The lower culture chamber of the reactor, having a much lower volume than that of the other culture chambers (from 10 to 1000 times smaller depending on the case) is used for the development of a very sample concentrate of a cell culture, which is introduced into this lower chamber through the axial passage 92 of the lower element 66. The fresh nutritional liquid circulating under the permeable wall 84 of the lower element 66 contains growth factors appropriate, to significantly increase the number of cells. The various culture stages of the column reactor can then be seeded one after the other, by opening the selective sealing means 96 and 98 provided at the upper end of the axial passage 92 of the upper element 66 and at the 'upper end of the duct 70.
Lorsque tous les étages de cultures ont été ainsi ensemencés, on peut par exemple les alimenter par un liquide nutritionnel ne contenant plus de facteurs de croissance, lorsque le réacteur est essentiellement des¬ tiné à la production de métabolites. Les métabolites ainsi que le liquide nutritionnel usé sortent de chaque étage de culture par le passage 90 de l'élément 66 cor¬ respondant et sont collectés à l'extrémité inférieure du conduit vertical 74.When all the stages of cultures have been seeded in this way, they can for example be fed with a nutritional liquid no longer containing growth factors, when the reactor is essentially intended for the production of metabolites. The metabolites and the spent nutritional liquid leave each culture stage through the passage 90 of the corresponding element 66 and are collected at the lower end of the vertical duct 74.
Les éléments constitutifs du réacteur colonne sont soit métalliques (par exemple en acier inoxydable) pour les éléments annulaires 66, soit en matière plas¬ tique (par exemple en silicone) pour les éléments annu- laires 68. Ces éléments peuvent être assemblés de façon étanche par simple compression de joints annulaires d'étanchéité, qui peuvent éventuellement être formés en saillie sur les faces opposées des éléments 68 en sili¬ cone. Le nettoyage et la stérilisation de ce réac¬ teur colonne se font sans difficulté, par exemple par circulation de vapeur d'eau dans les conduits 70 et 72, ainsi que dans les passages axiaux 92 des éléments 66. De plus, le réacteur colonne est entièrement démontable et donc facilement nettoyable.The constituent elements of the column reactor are either metallic (for example in stainless steel) for the annular elements 66, or in plas¬ tic material (for example in silicone) for the annular elements 68. These elements can be tightly assembled by simple compression of annular seals, which may optionally be formed projecting on the opposite faces of the elements 68 in sili¬ cone. The cleaning and sterilization of this column reactor is carried out without difficulty, for example by circulation of water vapor in the conduits 70 and 72, as well as in the axial passages 92 of the elements 66. In addition, the column reactor is fully removable and therefore easily cleanable.
De plus, le décolmatage des parois perméables selon l'invention peut être réalisé très simplement par des chocs thermiques, des chocs mécaniques, des vibra¬ tions, ou encore par magnéto-striction. In addition, the unclogging of the permeable walls according to the invention can be carried out very simply by thermal shocks, mechanical shocks, vibra¬ tions, or by magneto-necking.

Claims

REVENDICATIONS
1. Dispositif de culture de cellules ou de production de métabolites, comprenant au moins une chambre de culture cellulaire en milieu liquide dans la- quelle sont prévues deux parois (16, 84) sélectivement perméables, respectivement d'introduction de liquide nu¬ tritionnel frais dans la chambre de culture et de sortie de liquide nutritionnel usé de cette chambre, et des moyens (18, 84) d'alimentation en liquide nutritionnel frais sous pression, caractérisé en ce que les moyens (18, 84) d'alimentation comportent des surfaces sen¬ siblement parallèles et adjacentes, rigides et non per¬ méables, qui délimitent entre elles des micro-passages formant un distributeur à débit uniforme de liquide nu- tritionnel dans la chambre de culture.1. Device for cell culture or metabolite production, comprising at least one cell culture chamber in liquid medium in which two walls (16, 84) are provided which are selectively permeable, respectively for introducing fresh nu¬ tritional liquid in the culture chamber and the outlet for spent nutritional liquid from this chamber, and means (18, 84) for supplying fresh nutritional liquid under pressure, characterized in that the supply means (18, 84) comprise substantially parallel and adjacent, rigid and non-permeable surfaces which delimit between them micro-passages forming a distributor with a uniform flow rate of nutritional liquid in the culture chamber.
2. Dispositif selon la revendication 1, carac¬ térisé en ce que les micro-passages sont usinés dans les¬ dites surfaces qui sont appliquées les unes sur les autres. 2. Device according to claim 1, charac¬ terized in that the micro-passages are machined in les¬ said surfaces which are applied to each other.
3. Dispositif selon l'une des revendications précédentes, caractérisé en ce que lesdites surfaces dé¬ limitant les micro-passages ont un état de surface cali¬ bré et sont appliquées les unes sur les autres.3. Device according to one of the preceding claims, characterized in that said surfaces de¬ limiting micro-passages have a cali¬ bré surface condition and are applied to each other.
4. Dispositif selon l'une des revendications précédentes, caractérisé en ce que lesdites surfaces sont des faces adjacentes d'éléments (20,44) compacts et non poreux, qui sont empilés ou juxtaposés, ou imbriqués les uns dans les autres.4. Device according to one of the preceding claims, characterized in that said surfaces are adjacent faces of elements (20,44) compact and non-porous, which are stacked or juxtaposed, or nested one inside the other.
5. Dispositif selon l'une des revendications précédentes, caractérisé en ce que lesdites surfaces sont formées par les faces d'un ruban (56,84) enroulé à spires jointives et dont les extrémités sont fixes.5. Device according to one of the preceding claims, characterized in that said surfaces are formed by the faces of a ribbon (56,84) wound with contiguous turns and whose ends are fixed.
6. Dispositif selon la revendication 5, carac¬ térisé en ce que ledit ruban (56,84) enroulé à spires jointives est logé dans une cavité ou cuvette annulaire d'un élément de support à moyeu central (62,82) sur le- quel est fixée une extrémité du ruban, l'autre extrémité de ce dernier étant fixée sur la paroi périphérique de la cavité ou cuvette.6. Device according to claim 5, charac¬ terized in that said ribbon (56,84) wound with contiguous turns is housed in an annular cavity or bowl of a support element with central hub (62,82) on the- which one end of the ribbon is fixed, the other end of the latter being fixed to the peripheral wall of the cavity or bowl.
7. Dispositif selon la revendication 6, carac- térisé en ce que deux conduits (88) de circulation de li¬ quide nutritionnel débouchent dans le fond de ladite cu¬ vette (80) , l'un au voisinage de la périphérie de celle- ci et l'autre au voisinage du moyeu central (82) , un es¬ pace libre de passage de liquide nutritionnel étant mé- nagé entre le fond de la cuvette (80) et le ruban (84) .7. Device according to claim 6, characterized in that two conduits (88) for circulating nutritional liquid open into the bottom of said cup (80), one in the vicinity of the periphery thereof. Ci and the other in the vicinity of the central hub (82), a free space for the passage of nutritional liquid being arranged between the bottom of the bowl (80) and the ribbon (84).
8. Dispositif selon l'une des revendications précédentes, caractérisé en ce que lesdits moyens (84) d'alimentation en liquide nutritionnel frais constituent la paroi perméable d'introduction dans la chambre de cul- ture.8. Device according to one of the preceding claims, characterized in that said means (84) for supplying fresh nutritional liquid constitute the permeable wall for introduction into the culture chamber.
9. Dispositif selon la revendication 8, carac¬ térisé en ce que la paroi perméable de sortie de liquide nutritionnel usé a sensiblement la même structure que la¬ dite paroi d'introduction du liquide nutritionnel frais. 9. Device according to claim 8, charac¬ terized in that the permeable wall for the outlet of spent nutritional liquid has substantially the same structure as la¬ said wall for introducing the fresh nutritional liquid.
10. Dispositif selon l'une des revendications précédentes, caractérisé en ce qu'il comprend au moins deux chambres de culture cellulaire, verticalement super¬ posées et reliées l'une à l'autre, constituées par empi¬ lage d'éléments annulaires (66,68) alternés dont les uns forment les chambres de culture et dont les autres por¬ tent les parois perméables (84) précitées d'introduction et de sortie de liquide nutritionnel.10. Device according to one of the preceding claims, characterized in that it comprises at least two cell culture chambers, vertically super¬ posed and connected to each other, formed by empi¬ lage of annular elements ( 66,68) alternating, some of which form the culture chambers and the others of which carry the permeable walls (84) mentioned above for introducing and leaving nutritional liquid.
11. Dispositif selon la revendication 10, ca¬ ractérisé en ce que l'empilage d'éléments annulaires com- prend des conduits verticaux (70,72,74,76,78) pour l'alimentation en parallèle des chambres de culture en gaz et en liquide nutritionnel et la récupération du li¬ quide nutritionnel usé, ces conduits verticaux étant for¬ més par des orifices traversants axialement alignés des- dits éléments annulaires (66,68) .11. Device according to claim 10, ca¬ characterized in that the stacking of annular elements comprises vertical conduits (70,72,74,76,78) for the parallel supply of gas culture chambers and in nutritional liquid and the recovery of spent nutritional liquid, these vertical conduits being formed by axially aligned through orifices of said annular elements (66,68).
12. Dispositif selon la revendication 10 ou 11, caractérisé en ce qu'au moins un élément annulaire (66) intermédiaire comprend une paroi perméable (84) du type précité sur chacune de ses deux faces opposées, respectivement pour ,.1'introduction de liquide nutrition- nel frais dans une chambre de culture située sur une des faces de cet élément annulaire intermédiaire, et pour la sortie de liquide nutritionnel usé d'une chambre de cul¬ ture située sur l'autre face de cet élément intermé¬ diaire. 12. Device according to claim 10 or 11, characterized in that at least one intermediate annular element (66) comprises a permeable wall (84) of the aforementioned type on each of its two opposite faces, respectively, for the introduction of fresh nutritional liquid into a chamber culture located on one side of this intermediate annular element, and for the outlet of spent nutritional liquid from a cul¬ ture chamber located on the other face of this intermediate element.
13. Dispositif selon l'une des revendications13. Device according to one of claims
10 à 12, caractérisé en ce que chaque élément annulaire (68) formant une chambre de culture comprend un faisceau de tubes capillaires (94) à parois perméables aux gaz, traversant la chambre de culture et débouchant à leurs extrémités dans des orifices traversants (76,78) dudit élément annulaire (68) .10 to 12, characterized in that each annular element (68) forming a culture chamber comprises a bundle of capillary tubes (94) with walls permeable to gases, passing through the culture chamber and opening at their ends in through orifices (76 , 78) of said annular element (68).
14. Dispositif selon l'une des revendications 10 à 13, caractérisé en ce que les éléments annulaires (66) portant les parois perméables comprennent chacun un moyeu central (82) formé avec un passage axial traversant (92) de communication entre deux chambres de culture.14. Device according to one of claims 10 to 13, characterized in that the annular elements (66) carrying the permeable walls each comprise a central hub (82) formed with an axial through passage (92) for communication between two chambers culture.
15. Dispositif selon l'une des revendications 10 à 14, caractérisé en ce que la chambre de culture inférieure de ce dispositif a un volume très inférieur à celui des autres chambres de culture. 15. Device according to one of claims 10 to 14, characterized in that the lower culture chamber of this device has a volume much lower than that of the other culture chambers.
EP93913107A 1992-06-17 1993-06-10 Cell culture or metabolite production device Ceased EP0672112A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR9207345A FR2692589B1 (en) 1992-06-17 1992-06-17 DEVICE FOR CULTURING CELLS OR PRODUCING METABOLITES.
FR9207345 1992-06-17
PCT/FR1993/000551 WO1993025658A1 (en) 1992-06-17 1993-06-10 Cell culture or metabolite production device

Publications (1)

Publication Number Publication Date
EP0672112A1 true EP0672112A1 (en) 1995-09-20

Family

ID=9430836

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93913107A Ceased EP0672112A1 (en) 1992-06-17 1993-06-10 Cell culture or metabolite production device

Country Status (3)

Country Link
EP (1) EP0672112A1 (en)
FR (1) FR2692589B1 (en)
WO (1) WO1993025658A1 (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4201845A (en) * 1976-04-12 1980-05-06 Monsanto Company Cell culture reactor
US5068195A (en) * 1988-11-02 1991-11-26 Du Pont Merck Pharmaceutical Company Dot matrix membrane cell expansion and maintenance vessel

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9325658A1 *

Also Published As

Publication number Publication date
FR2692589B1 (en) 1995-06-23
FR2692589A1 (en) 1993-12-24
WO1993025658A1 (en) 1993-12-23

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