EP1507846A1 - Cell culture device - Google Patents
Cell culture deviceInfo
- Publication number
- EP1507846A1 EP1507846A1 EP03752829A EP03752829A EP1507846A1 EP 1507846 A1 EP1507846 A1 EP 1507846A1 EP 03752829 A EP03752829 A EP 03752829A EP 03752829 A EP03752829 A EP 03752829A EP 1507846 A1 EP1507846 A1 EP 1507846A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- liquid
- enclosure
- mouth
- cells
- air
- 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.)
- Withdrawn
Links
- 238000004113 cell culture Methods 0.000 title claims description 9
- 239000007788 liquid Substances 0.000 claims abstract description 80
- 238000002347 injection Methods 0.000 claims abstract description 7
- 239000007924 injection Substances 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 31
- 239000002609 medium Substances 0.000 description 16
- 239000001963 growth medium Substances 0.000 description 8
- 150000002500 ions Chemical class 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 230000005499 meniscus Effects 0.000 description 5
- 230000004913 activation Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000005684 electric field Effects 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 210000000287 oocyte Anatomy 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 239000002120 nanofilm Substances 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 150000001669 calcium Chemical class 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000004078 waterproofing Methods 0.000 description 1
Classifications
-
- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
-
- 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
Definitions
- the present invention relates to a cell culture device allowing their treatment by different media while avoiding their manipulation.
- the document FR 2 659 347 (published on 13/151991) describes a device for the culture of cells comprising an enclosure intended to receive the cells.
- the enclosure comprises a horizontal support formed by two glass plates juxtaposed and spaced so as to form between them a slot having a width less than the diameters of the cells.
- the cells to be treated are positioned on the slot.
- This enclosure is intended to contain liquid culture or pulse media.
- the various liquid media are successively injected into the enclosure by separate tubes positioned above the wall supporting the cells. Part of the liquid contained injected into the enclosure is evacuated by one or more tubing (s) located (s) below the wall. Another part of this liquid is evacuated by overflow.
- the cells are held on the slit by virtue of the depression caused by the aspiration of the liquid medium by the evacuation tube (s).
- a device is intended in particular for the culture of oocytes, fertilized eggs or embryos, etc.
- a culture medium is injected into the enclosure and the cells to be activated are positioned in this culture medium.
- the culture medium rich in ions is then removed and simultaneously replaced by an impulse medium containing Ca 2+ ions.
- the cells are subjected to an electric field pulse which causes the transient electro-waterproofing of their plasma membrane and the penetration of Ca 2+ ions inside. cells.
- the impulse medium is in turn evacuated and replaced by the culture medium.
- An advantage of this device is that it allows the treatment of cells by different media while avoiding their manipulation.
- a problem posed by this type of device is that the replacement of one medium by another is relatively long, which limits the frequency of alternation of the media in the enclosure.
- a minimum time of injection of the pulse medium is necessary (of the order of 40 seconds) to replace the culture medium with the medium d 'impulse. This minimum time indeed guarantees sufficient washing of the cells by the pulse medium.
- the pulse medium would contain residual ions from the culture medium. During the application of the electric field, these ions would induce a trans-membrane ion current which would cause the destruction of cells.
- Another problem with washing is that prolonged exposure of cells in the pulse medium with low ionic strength disturbs the balance of the cells and exposes them to deleterious effects. To preserve cell survival, it is therefore necessary to reduce the cell washing time.
- An object of the invention is to provide a cell culture device making it possible to quickly replace the medium in which the cells are placed.
- the cells tend to translate along the slit of the support element towards the center of the chamber where they group. They are compressed against each other. Reducing the space between cells changes the efficiency of washing at the periphery of each cell.
- these films can constitute conductivity bridges between the electrodes. These conductivity bridges establish a short circuit between the electrodes. This results in a significant decrease in the efficiency of the applied electric field.
- the invention provides a device intended in particular for cell culture comprising an enclosure intended to receive the cells, said enclosure comprising at least one liquid injection duct, the surface of the liquid contained in the enclosure being in free contact with air, characterized in that it comprises a collecting element capable of establishing with the free surface of the liquid at least one liquid bridge, the collecting element comprising a mouthpiece positioned substantially at the level of the free surface of the liquid contained in the enclosure, the mouth being maintained in depression so that it absorbs a flow of air which drives the surface film of the liquid via the liquid bridge.
- the device of the invention makes it possible to create a liquid bridge in the form of a meniscus between the free surface of the liquid and the collecting element. This meniscus regulates by surface balance the surface tensions on the surface of the liquid contained in the enclosure.
- Such a device makes it possible, thanks to the air flow, to continuously eliminate the excess liquid in the enclosure and thus to maintain a constant level of liquid.
- the collecting element is positioned on a rim of a wall of the enclosure, its mouth being set back from the wall.
- a portion of the rim located in front of the mouth of the collecting element is covered with a hydrophilic substance.
- the mouth is set back from the wall at a distance d of between 10 and 30 ⁇ m.
- the mouth of the manifold has an elongated shape and extends longitudinally along the edge of the wall.
- the opening has a length of the order of 2 to 4 mm. In one implementation of the invention, the opening has a height of the order of 0.15 to 0.30 mm.
- the mouth of the collecting element has a generally rectangular shape.
- the device comprises several elements positioned along the edges of the walls of the enclosure.
- the invention also relates to a method for regulating the level of a liquid contained in an enclosure intended to receive cells, said enclosure comprising at least one liquid injection duct, the surface of the liquid contained in the enclosure being in free contact with the air, characterized in that at least one liquid bridge is established with the free surface of the liquid and an air flow is sucked near the liquid bridge, so that this air flow carries the surface film of the liquid through the liquid bridge.
- FIG. 1 is a diagram representative of an example of a cell culture device in accordance with an implementation of the invention
- FIG. 2 is a detailed diagram of a collector of the device of FIG. 1,
- FIG. 1 is a sectional diagram of the collector of Figure 2.
- the cell culture device shown comprises an enclosure 100 comprising walls 11, 12, 13 defining a chamber 20 intended to contain a liquid medium .
- the chamber is filled with a liquid medium corresponding to the treatment phase of the cells in progress.
- a horizontal support element is positioned, formed by the juxtaposition of two glass plates 101 and 102.
- the glass plates 101 and 102 are kept embedded in the side walls 11 and 12 of the enclosure. These glass plates 101 and 102 are spaced so as to define between them a rectilinear slot 103 of width less than the diameter of the oocytes 10 to be treated.
- the enclosure 100 also comprises electrodes 111 and 112 extending longitudinally on either side of the slot 103.
- the culture liquid is brought into the upper part of the chamber
- the free surface of the liquid contained in the enclosure 100 forms a molecular film 30 having a convex shape.
- This film 30 is made up of an alignment of oriented molecules whose hydrophilic function is positioned towards the fluid and the hydrophobic portion is positioned towards the outside air.
- the culture device comprises collectors 40 positioned on a horizontal rim 21 of the wall 11 of the chamber 20.
- the device may include collectors positioned on flanges 22 or 23 of the walls 12 or 13 (at the locations shown in dotted lines).
- These collectors 40 can be arranged at regular intervals around the chamber 20.
- FIG 2 is a diagram showing in more detail a collector 40 of the cell culture device of Figure 1.
- Two liquid bridges 51 and 52 are formed between the surface 30 of the liquid contained in the chamber 20 and the vertical edges of the opening 42. These liquid bridges 51 and 52 are formed by deformations of the surface 30 of the liquid (or menisci) in contact with the edges of the opening 42.
- the collector 40 also comprises a discharge duct 43 connected to a suction device, not shown.
- This suction device makes it possible to aspirate the air contained in the manifold 40.
- This suction causes air circulation through the opening 42 which drives an upper portion of the liquid contained in the enclosure via the liquid bridges 51 and 52.
- the molecular film 30 on the surface of the liquid is permanently entrained by the air circulating in the collector and removed from the surface of the liquid.
- the length L of the opening has been chosen so as to be at least twice the capillary length k "1 of the liquid contained inside the enclosure. This characteristic guarantees the formation of two lateral menisci and therefore the non-sealing of the opening 42 by the liquid. The air is therefore always free to circulate through the opening 42.
- the liquids generally used for the treatment of cells generally have capillary lengths between 1 mm and 2 mm.
- the length L of the opening will therefore preferably be of the order of 2 to 4mm.
- the collector 40 is positioned set back from the wall by a distance d. Indeed, the opening must not be too close to the wall 11, in which case, the capillary forces would lead to the formation of a single liquid bridge which would close the opening 42 and which would empty the chamber 20 of the liquid which it contains.
- the opening 42 must not be too far from the wall 11, in which case the manifold would no longer have any effect on the liquid. Furthermore, a distance d of a few microns leads to the formation of a concave meniscus.
- the concave shape has several drawbacks:
- the concave shape modifies the optical path of the light and makes it more difficult for the operator to view the cells.
- the distance d is preferably between 10 and 30 ⁇ m.
- the distance d is 20 ⁇ m.
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- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
DISPOSITIF DE CULTURE DE CELLULES CELL CULTURE DEVICE
La présente invention concerne un dispositif de culture de cellules permettant leur traitement par différents milieux en évitant leur manipulation. Le document FR 2 659 347 (publié le 13/09/1991) décrit un dispositif pour la culture de cellules comportant une enceinte destinée à recevoir les cellules. L'enceinte comprend un support horizontal formé par deux plaques de verre juxtaposées et espacées de manière à former entre elles une fente présentant une largeur inférieure aux diamètres des cellules. Les cellules à traiter sont positionnées sur la fente. Cette enceinte est destinée à contenir des milieux liquides de culture ou d'impulsion. Les différents milieux liquides sont injectés successivement dans l'enceinte par des tubulures distinctes positionnées au dessus de la paroi supportant les cellules. Une partie du liquide contenu injecté dans l'enceinte est évacuée par une ou plusieurs tubulure(s) située(s) au-dessous de la paroi. Une autre partie de ce liquide est évacuée par débordement.The present invention relates to a cell culture device allowing their treatment by different media while avoiding their manipulation. The document FR 2 659 347 (published on 13/09/1991) describes a device for the culture of cells comprising an enclosure intended to receive the cells. The enclosure comprises a horizontal support formed by two glass plates juxtaposed and spaced so as to form between them a slot having a width less than the diameters of the cells. The cells to be treated are positioned on the slot. This enclosure is intended to contain liquid culture or pulse media. The various liquid media are successively injected into the enclosure by separate tubes positioned above the wall supporting the cells. Part of the liquid contained injected into the enclosure is evacuated by one or more tubing (s) located (s) below the wall. Another part of this liquid is evacuated by overflow.
Dans ce dispositif, les cellules sont maintenues sur la fente grâce à la dépression provoquée par l'aspiration du milieu liquide par la ou les tubulure(s) d'évacuation. Un tel dispositif est destiné notamment à la culture d'ovocytes, d'œufs fécondés ou d'embryons, etc.In this device, the cells are held on the slit by virtue of the depression caused by the aspiration of the liquid medium by the evacuation tube (s). Such a device is intended in particular for the culture of oocytes, fertilized eggs or embryos, etc.
En particulier, il peut être utilisé pour l'activation d'ovocytes expérimentaux, cette activation étant nécessaire pour le bon développement ultérieur des embryons. Pour provoquer cette activation, on injecte dans l'enceinte un milieu de culture et on positionne les cellules à activer dans ce milieu de culture. Le milieu de culture riche en ions est ensuite évacué et simultanément remplacé par un milieu d'impulsion contenant des ions Ca2+. Lorsque le milieu de culture a totalement été évacué et remplacé par le milieu d'impulsion, on soumet les cellules à une impulsion de champ électrique qui provoque l'electroperméabilisation transitoire de leur membrane plasmique et la pénétration des ions Ca2+ à l'intérieur des cellules. Puis le milieu d'impulsion est à son tour évacué et remplacé par le milieu de culture. Ces étapes sont répétées un certain nombre de fois de sorte que les cellules sont soumises à une série contrôlée d'impulsions calciques qui déclenche leur activation.In particular, it can be used for the activation of experimental oocytes, this activation being necessary for the good subsequent development of the embryos. To cause this activation, a culture medium is injected into the enclosure and the cells to be activated are positioned in this culture medium. The culture medium rich in ions is then removed and simultaneously replaced by an impulse medium containing Ca 2+ ions. When the culture medium has been completely removed and replaced by the pulse medium, the cells are subjected to an electric field pulse which causes the transient electro-waterproofing of their plasma membrane and the penetration of Ca 2+ ions inside. cells. Then the impulse medium is in turn evacuated and replaced by the culture medium. These steps are repeated a certain number of times so that cells are subjected to a controlled series of calcium pulses that trigger their activation.
Un avantage de ce dispositif est qu'il permet le traitement des cellules par différents milieux en évitant leur manipulation. Toutefois, un problème posé par ce type de dispositif est que le remplacement d'un milieu par un autre est relativement long, ce qui limite la fréquence d'alternance des milieux dans l'enceinte.An advantage of this device is that it allows the treatment of cells by different media while avoiding their manipulation. However, a problem posed by this type of device is that the replacement of one medium by another is relatively long, which limits the frequency of alternation of the media in the enclosure.
Par exemple, dans le cas où le dispositif est utilisé pour l'activation de cellules, un temps minimal d'injection du milieu d'impulsion est nécessaire (de l'ordre de 40 secondes) pour remplacer le milieu de culture par le milieu d'impulsion. Ce temps minimal garantit en effet un lavage suffisant des cellules par le milieu d'impulsion.For example, in the case where the device is used for the activation of cells, a minimum time of injection of the pulse medium is necessary (of the order of 40 seconds) to replace the culture medium with the medium d 'impulse. This minimum time indeed guarantees sufficient washing of the cells by the pulse medium.
Dans le cas où ce temps minimal de lavage ne serait pas respecté, le milieu d'impulsion contiendrait des ions résiduels provenant du milieu de culture. Lors de l'application du champ électrique, ces ions induiraient un courant ionique trans-membranaire qui provoquerait la destruction des cellules.In the event that this minimum washing time is not observed, the pulse medium would contain residual ions from the culture medium. During the application of the electric field, these ions would induce a trans-membrane ion current which would cause the destruction of cells.
Un autre problème lié au lavage est que l'exposition prolongée des cellules dans le milieu d'impulsion présentant une faible force ionique perturbe l'équilibre des cellules et les expose à des effets délétères. Pour préserver la survie cellulaire, il est donc nécessaire de réduire la durée de lavage des cellules.Another problem with washing is that prolonged exposure of cells in the pulse medium with low ionic strength disturbs the balance of the cells and exposes them to deleterious effects. To preserve cell survival, it is therefore necessary to reduce the cell washing time.
Un but de l'invention est de fournir un dispositif de culture de cellules permettant de remplacer rapidement le milieu dans lequel les cellules sont placées.An object of the invention is to provide a cell culture device making it possible to quickly replace the medium in which the cells are placed.
On a remarqué que l'évacuation du liquide par débordement est un phénomène irrégulier. En effet, pendant l'injection du liquide dans l'enceinte, la surface libre du liquide s'élève au-dessus du plan supérieur de la chambre de telle sorte que la surface du liquide prend la forme d'un ménisque convexe. Ce phénomène est lié aux tensions superficielles qui s'exercent à la surface du liquide et à la mouillabilité des parois verticales de la chambre par ce liquide. La dénivellation du liquide au-dessus des bords de la chambre doit atteindre une valeur critique pour que l'équilibre entre les forces liées aux tensions superficielles d'une part et les forces liées à la gravité d'autre part, soit rompu. Cette rupture entraîne l'écoulement du surplus de liquide par dessus les parois de la chambre.It has been noted that the evacuation of the liquid by overflow is an irregular phenomenon. In fact, during the injection of the liquid into the enclosure, the free surface of the liquid rises above the upper plane of the chamber so that the surface of the liquid takes the form of a convex meniscus. This phenomenon is linked to the surface tensions which are exerted on the surface of the liquid and to the wettability of the vertical walls of the chamber by this liquid. The difference in level of the liquid above the edges of the chamber must reach a critical value so that the equilibrium between the forces linked to surface tensions on the one hand and the forces linked to gravity on the other hand, is broken. This rupture causes the excess liquid to flow over the walls of the chamber.
Il en résulte que l'évacuation du liquide par débordement se produit de manière discontinue et imprévisible. Notamment, il peut arriver que durant une phase de remplacement d'un milieu par un autre, le milieu à remplacer ne soit pas évacué. Cette évacuation n'est donc pas satisfaisante car elle ne garantit pas le lavage des cellules lors de cette phase de remplacement. Par ailleurs, un autre problème est que cet écoulement discontinu génère une succession d'ondes de choc dans le liquide. Ces ondes de choc déplacent les cellules sur le support.As a result, the evacuation of the liquid by overflow occurs discontinuously and unpredictably. In particular, it may happen that during a phase of replacement of one medium by another, the medium to be replaced is not removed. This evacuation is therefore not satisfactory because it does not guarantee washing of the cells during this replacement phase. Another problem is that this discontinuous flow generates a succession of shock waves in the liquid. These shock waves move the cells on the support.
Les cellules ont tendance à se translater le long de la fente de l'élément support vers le centre de la chambre où elles de regroupent. Elles sont comprimées les unes contre les autres. La réduction de l'espace entre les cellules modifie l'efficacité du lavage à la périphérie de chaque cellule.The cells tend to translate along the slit of the support element towards the center of the chamber where they group. They are compressed against each other. Reducing the space between cells changes the efficiency of washing at the periphery of each cell.
Enfin, un autre problème encore est que l'interface entre le milieu de culture et le gaz environnant est le lieu de phénomènes de surface. Des films atomiques ou moléculaires se forment à la surface libre du liquide. Ces films ne sont pas renouvelés lors du remplacement d'un milieu par un autre dans l'enceinte.Finally, yet another problem is that the interface between the culture medium and the surrounding gas is the site of surface phenomena. Atomic or molecular films form on the free surface of the liquid. These films are not renewed when a medium is replaced by another in the enclosure.
Dans le cas où le dispositif de culture est utilisé pour appliquer aux cellules des impulsions de champ électrique, ces films peuvent constituer des ponts de conductivité entre les électrodes. Ces ponts de conductivité établissent un court-circuit entre les électrodes. Il en résulte une diminution significative de l'efficacité du champ électrique appliqué.In the case where the culture device is used to apply electric field pulses to the cells, these films can constitute conductivity bridges between the electrodes. These conductivity bridges establish a short circuit between the electrodes. This results in a significant decrease in the efficiency of the applied electric field.
Pour pallier ces inconvénients, l'invention propose un dispositif destiné notamment à la culture de cellules comprenant une enceinte destinée à recevoir les cellules, ladite enceinte comportant au moins un conduit d'injection de liquide, la surface du liquide contenu dans l'enceinte étant en contact libre avec l'air, caractérisé en ce qu'il comprend un élément collecteur apte à établir avec la surface libre du liquide au moins un pont liquide, l'élément collecteur comprenant une embouchure positionnée sensiblement au niveau de la surface libre du liquide contenu dans l'enceinte, l'embouchure étant maintenue en dépression de sorte qu'elle absorbe un flux d'air qui entraîne le film de surface du liquide via le pont liquide. Le dispositif de l'invention permet de créer un pont liquide sous la forme d'un ménisque entre la surface libre du liquide et l'élément collecteur. Ce ménisque régule par effet d'équilibre les tensions superficielles à la surface du liquide contenu dans l'enceinte.To overcome these drawbacks, the invention provides a device intended in particular for cell culture comprising an enclosure intended to receive the cells, said enclosure comprising at least one liquid injection duct, the surface of the liquid contained in the enclosure being in free contact with air, characterized in that it comprises a collecting element capable of establishing with the free surface of the liquid at least one liquid bridge, the collecting element comprising a mouthpiece positioned substantially at the level of the free surface of the liquid contained in the enclosure, the mouth being maintained in depression so that it absorbs a flow of air which drives the surface film of the liquid via the liquid bridge. The device of the invention makes it possible to create a liquid bridge in the form of a meniscus between the free surface of the liquid and the collecting element. This meniscus regulates by surface balance the surface tensions on the surface of the liquid contained in the enclosure.
Un tel dispositif permet grâce au flux d'air, d'éliminer de manière continue le surplus de liquide dans l'enceinte et ainsi de maintenir un niveau de liquide constant.Such a device makes it possible, thanks to the air flow, to continuously eliminate the excess liquid in the enclosure and thus to maintain a constant level of liquid.
Il permet également d'obtenir un renouvellement permanent du film de surface du liquide de l'enceinte.It also makes it possible to obtain a permanent renewal of the surface film of the enclosure liquid.
Dans une mise en oeuvre de l'invention, l'élément collecteur est positionné sur un rebord d'une paroi de l'enceinte, son embouchure étant en retrait de la paroi.In one implementation of the invention, the collecting element is positioned on a rim of a wall of the enclosure, its mouth being set back from the wall.
De préférence, une portion du rebord située devant l'embouchure de l'élément collecteur est recouverte d'une substance hydrophile.Preferably, a portion of the rim located in front of the mouth of the collecting element is covered with a hydrophilic substance.
De préférence, l'embouchure est en retrait de la paroi à une distance d comprise entre 10 et 30μm.Preferably, the mouth is set back from the wall at a distance d of between 10 and 30 μm.
De préférence, l'embouchure du collecteur présente une forme allongée et s'étend longitudinalement le long du bord de la paroi.Preferably, the mouth of the manifold has an elongated shape and extends longitudinally along the edge of the wall.
Dans une mise en œuvre de l'invention, l'ouverture présente une longueur de l'ordre de 2 à 4 mm. Dans une mise en œuvre de l'invention, l'ouverture présente une hauteur de l'ordre de 0,15 à 0,30 mm.In one implementation of the invention, the opening has a length of the order of 2 to 4 mm. In one implementation of the invention, the opening has a height of the order of 0.15 to 0.30 mm.
Dans une mise en œuvre de l'invention, l'embouchure de l'élément collecteur présente une forme générale rectangulaire.In one implementation of the invention, the mouth of the collecting element has a generally rectangular shape.
Avantageusement, le dispositif comprend plusieurs éléments positionnés le long des bords des parois de l'enceinte.Advantageously, the device comprises several elements positioned along the edges of the walls of the enclosure.
L'invention concerne également un procédé de régulation du niveau d'un liquide contenu dans une enceinte destinée à recevoir des cellules, ladite enceinte comportant au moins un conduit d'injection de liquide, la surface du liquide contenu dans l'enceinte étant en contact libre avec l'air, caractérisé en ce qu'on établit avec la surface libre du liquide au moins un pont liquide et on aspire un flux d'air à proximité du pont liquide, de sorte que ce flux d'air entraîne le film de surface du liquide via le pont liquide. D'autres caractéristiques et avantages ressortiront encore de la description qui suit, laquelle est purement illustrative et non limitative et doit être lue en regard des figures annexées parmi lesquelles :The invention also relates to a method for regulating the level of a liquid contained in an enclosure intended to receive cells, said enclosure comprising at least one liquid injection duct, the surface of the liquid contained in the enclosure being in free contact with the air, characterized in that at least one liquid bridge is established with the free surface of the liquid and an air flow is sucked near the liquid bridge, so that this air flow carries the surface film of the liquid through the liquid bridge. Other characteristics and advantages will also emerge from the description which follows, which is purely illustrative and not limiting and should be read with reference to the appended figures among which:
- la figure 1 est un schéma représentatif d'un exemple de dispositif de culture de cellules conforme à une mise en œuvre de l'invention, - la figure 2 est un schéma de détail d'un collecteur du dispositif de la figure 1 ,FIG. 1 is a diagram representative of an example of a cell culture device in accordance with an implementation of the invention, FIG. 2 is a detailed diagram of a collector of the device of FIG. 1,
- la figure 3 est un schéma en coupe du collecteur de la figure 2. Sur la figure 1 , le dispositif de culture de cellules représenté comprend une enceinte 100 comprenant des parois 11 , 12, 13 délimitant une chambre 20 destinée à contenir un milieu liquide. La chambre est remplie d'un milieu liquide correspondant à la phase de traitement des cellules en cours. Dans la chambre 20, un élément support horizontal est positionné, formé par la juxtaposition de deux plaques de verre 101 et 102. Les plaques de verre 101 et 102 sont maintenues encastrées dans les parois 11 et 12 latérales de l'enceinte. Ces plaques de verre 101 et 102 sont espacées de manière à définir entre elles une fente rectiligne 103 de largeur inférieure au diamètre des ovocytes 10 à traiter.- Figure 3 is a sectional diagram of the collector of Figure 2. In Figure 1, the cell culture device shown comprises an enclosure 100 comprising walls 11, 12, 13 defining a chamber 20 intended to contain a liquid medium . The chamber is filled with a liquid medium corresponding to the treatment phase of the cells in progress. In the chamber 20, a horizontal support element is positioned, formed by the juxtaposition of two glass plates 101 and 102. The glass plates 101 and 102 are kept embedded in the side walls 11 and 12 of the enclosure. These glass plates 101 and 102 are spaced so as to define between them a rectilinear slot 103 of width less than the diameter of the oocytes 10 to be treated.
L'enceinte 100 comprend également des électrodes 111 et 112 s'étendant longitudinalement de part et d'autre de la fente 103. Le liquide de culture est amené en partie supérieure de la chambreThe enclosure 100 also comprises electrodes 111 and 112 extending longitudinally on either side of the slot 103. The culture liquid is brought into the upper part of the chamber
20 par une série de conduits dont les embouchures 121 , 122, 123, 124, 125, 126 sont positionnées à intervalles réguliers le long des parois 11 , 12 et 13. Par ailleurs, une partie du liquide contenu dans l'enceinte est évacué par un conduit d'évacuation 104 situé à un niveau inférieur à celui de l'élément support. Le courant de liquide maintient par dépression les ovocytes 10 plaqués sur la fente 103.20 by a series of conduits, the mouths 121, 122, 123, 124, 125, 126 of which are positioned at regular intervals along the walls 11, 12 and 13. Furthermore, part of the liquid contained in the enclosure is evacuated by an exhaust duct 104 located at a level lower than that of the support element. The current of liquid maintains by vacuum the oocytes 10 pressed against the slot 103.
La surface libre du liquide contenu dans l'enceinte 100 forme un film moléculaire 30 présentant une forme convexe. Ce film 30 est constitué par un alignement de molécules orientées dont la fonction hydrophile se positionne vers le fluide et la portion hydrophobe se positionne vers l'air extérieur.The free surface of the liquid contained in the enclosure 100 forms a molecular film 30 having a convex shape. This film 30 is made up of an alignment of oriented molecules whose hydrophilic function is positioned towards the fluid and the hydrophobic portion is positioned towards the outside air.
Pour éliminer le film moléculaire 30 et absorber le surplus de liquide contenu dans la chambre 20, le dispositif de culture comprend des collecteurs 40 positionnés sur un rebord horizontal 21 de la paroi 11 de la chambre 20.To eliminate the molecular film 30 and absorb the surplus liquid contained in the chamber 20, the culture device comprises collectors 40 positioned on a horizontal rim 21 of the wall 11 of the chamber 20.
De manière symétrique, le dispositif peut comprendre des collecteurs positionnés sur des rebords 22 ou 23 des parois 12 ou 13 (aux emplacements représentés en traits pointillés).Symmetrically, the device may include collectors positioned on flanges 22 or 23 of the walls 12 or 13 (at the locations shown in dotted lines).
Ces collecteurs 40 peuvent être disposés à intervalles réguliers autour de la chambre 20.These collectors 40 can be arranged at regular intervals around the chamber 20.
La figure 2 est un schéma représentant plus en détail un collecteur 40 du dispositif de culture de cellules de la figure 1. Ce collecteur 40 est positionné sur le rebord 21 de la paroi 11 de l'enceinte en retrait par rapport à cette paroi d'une distance d. Il comprend un corps 41 creux présentant une ouverture rectangulaire 42 s'étendant le long de la paroi 11. Cette ouverture présente une longueur L=2mm et une hauteur l=0,2mm. Deux ponts liquides 51 et 52 sont formés entre la surface 30 du liquide contenu dans la chambre 20 et les bords verticaux de l'ouverture 42. Ces ponts liquides 51 et 52 sont formés par des déformations de la surface 30 du liquide (ou ménisques) au contact des bords de l'ouverture 42.Figure 2 is a diagram showing in more detail a collector 40 of the cell culture device of Figure 1. This collector 40 is positioned on the flange 21 of the wall 11 of the enclosure recessed with respect to this wall of a distance d. It comprises a hollow body 41 having a rectangular opening 42 extending along the wall 11. This opening has a length L = 2mm and a height l = 0.2mm. Two liquid bridges 51 and 52 are formed between the surface 30 of the liquid contained in the chamber 20 and the vertical edges of the opening 42. These liquid bridges 51 and 52 are formed by deformations of the surface 30 of the liquid (or menisci) in contact with the edges of the opening 42.
Le collecteur 40 comprend par ailleurs un conduit d'évacuation 43 relié à un dispositif d'aspiration non représenté. Ce dispositif d'aspiration permet d'aspirer l'air contenu dans le collecteur 40. Cette aspiration provoque une circulation d'air à travers l'ouverture 42 qui entraîne une portion supérieure du liquide contenu dans l'enceinte via les ponts liquides 51 et 52. Il en résulte que le film moléculaire 30 à la surface du liquide est en permanence entraîné par l'air circulant dans le collecteur et éliminé de la surface du liquide.The collector 40 also comprises a discharge duct 43 connected to a suction device, not shown. This suction device makes it possible to aspirate the air contained in the manifold 40. This suction causes air circulation through the opening 42 which drives an upper portion of the liquid contained in the enclosure via the liquid bridges 51 and 52. As a result, the molecular film 30 on the surface of the liquid is permanently entrained by the air circulating in the collector and removed from the surface of the liquid.
La longueur L de l'ouverture a été choisie de manière à être au moins deux fois supérieure à la longueur capillaire k"1 du liquide contenu dans l'enceinte. Cette caractéristique garantit la formation de deux ménisques latéraux et donc la non obturation de l'ouverture 42 par le liquide. L'air est donc toujours libre de circuler à travers l'ouverture 42.The length L of the opening has been chosen so as to be at least twice the capillary length k "1 of the liquid contained inside the enclosure. This characteristic guarantees the formation of two lateral menisci and therefore the non-sealing of the opening 42 by the liquid. The air is therefore always free to circulate through the opening 42.
Les liquides généralement utilisés pour le traitement des cellules présentent généralement des longueurs capillaires comprises entre 1 mm et 2mm. La longueur L de l'ouverture sera donc de préférence de l'ordre de 2 à 4mm.The liquids generally used for the treatment of cells generally have capillary lengths between 1 mm and 2 mm. The length L of the opening will therefore preferably be of the order of 2 to 4mm.
Le collecteur 40 est positionné en retrait de la paroi d'une distance d. En effet, l'ouverture ne doit pas être trop proche de la paroi 11 , auquel cas, les forces de capillarité conduiraient à la formation d'un pont liquide unique qui obturerait l'ouverture 42 et qui viderait la chambre 20 du liquide qu'elle contient.The collector 40 is positioned set back from the wall by a distance d. Indeed, the opening must not be too close to the wall 11, in which case, the capillary forces would lead to the formation of a single liquid bridge which would close the opening 42 and which would empty the chamber 20 of the liquid which it contains.
Néanmoins, l'ouverture 42 ne doit pas être trop éloignée de la paroi 11 , auquel cas, le collecteur n'aurait plus aucun effet sur le liquide. Par ailleurs, une distance d de quelques microns conduit à la formation d'un ménisque concave. Or la forme concave présente plusieurs inconvénients :However, the opening 42 must not be too far from the wall 11, in which case the manifold would no longer have any effect on the liquid. Furthermore, a distance d of a few microns leads to the formation of a concave meniscus. However, the concave shape has several drawbacks:
- d'une part, elle augmente les tensions superficielles vers le fond de la chambre 20, au voisinage des cellules 10, ce qui rend la mise en place ou le prélèvement des cellules plus délicat pour l'opérateur,on the one hand, it increases the surface tensions towards the bottom of the chamber 20, in the vicinity of the cells 10, which makes the placement or removal of the cells more difficult for the operator,
- d'autre part, la forme concave modifie le trajet optique de la lumière et rend plus difficile la visualisation des cellules par l'opérateur.- on the other hand, the concave shape modifies the optical path of the light and makes it more difficult for the operator to view the cells.
Par conséquent, il est souhaitable de maintenir un ménisque de forme convexe stable. C'est pourquoi, la distance d est de préférence comprise entre 10 et 30μm.Therefore, it is desirable to maintain a meniscus of stable convex shape. This is why the distance d is preferably between 10 and 30 μm.
Ainsi, dans le collecteur 40 représenté sur la figure 2, la distance d est de 20μm.Thus, in the collector 40 shown in FIG. 2, the distance d is 20 μm.
De manière préférentielle, les rebords 21 , 22 et 23 des parois 11 ,Preferably, the edges 21, 22 and 23 of the walls 11,
12 et 13 de l'enceinte sont recouverts d'une substance hydrophobe, excepté devant les ouvertures 42 des collecteurs où ils sont recouverts d'une substance hydrophile. Cette disposition favorise l'amorce de pont liquide au niveau des collecteurs 40. Sur la figure 3, un collecteur 40 est présenté en coupe le long du conduit d'évacuation 43. Le trajet de l'air aspiré est représenté par des flèches. 12 and 13 of the enclosure are covered with a hydrophobic substance, except in front of the openings 42 of the collectors where they are covered with a hydrophilic substance. This arrangement favors the initiation of a liquid bridge at the level of the collectors 40. In Figure 3, a manifold 40 is presented in section along the exhaust duct 43. The path of the aspirated air is represented by arrows.
Claims
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0206185A FR2839979B1 (en) | 2002-05-21 | 2002-05-21 | CELL CULTURE DEVICE |
| FR0206185 | 2002-05-21 | ||
| PCT/FR2003/001496 WO2003097787A1 (en) | 2002-05-21 | 2003-05-16 | Cell culture device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1507846A1 true EP1507846A1 (en) | 2005-02-23 |
Family
ID=29414954
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP03752829A Withdrawn EP1507846A1 (en) | 2002-05-21 | 2003-05-16 | Cell culture device |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US7527965B2 (en) |
| EP (1) | EP1507846A1 (en) |
| JP (1) | JP4410848B2 (en) |
| AU (1) | AU2003260552A1 (en) |
| FR (1) | FR2839979B1 (en) |
| WO (1) | WO2003097787A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1851304A4 (en) * | 2005-02-23 | 2009-10-21 | Cook William A Australia | CULTURE DEVICE |
| CN102124094A (en) * | 2008-07-16 | 2011-07-13 | 以库克泌尿外科公司为商用名的万斯产品公司 | Micro-fluidic cell manipulation and holding device |
| US10494626B2 (en) * | 2010-05-12 | 2019-12-03 | Cellectis S.A. | Dynamic mixing and electroporation chamber and system |
| EP3504315A4 (en) | 2016-08-27 | 2020-04-15 | 3D Biotek, LLC | Bioreactor |
| CN110190189B (en) * | 2019-06-06 | 2020-08-04 | 河南大学 | Fiber liquid bridge film preparation device |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2078138A1 (en) * | 1990-03-12 | 1991-09-13 | Jean-Pierre Ozil | Artificial stimulation process of cells and ovocytes culture device |
| US6423536B1 (en) * | 1999-08-02 | 2002-07-23 | Molecular Dynamics, Inc. | Low volume chemical and biochemical reaction system |
| DE19948473A1 (en) * | 1999-10-08 | 2001-04-12 | Nmi Univ Tuebingen | Method and device for measuring cells in a liquid environment |
| US6699697B2 (en) * | 2000-02-11 | 2004-03-02 | Yale University | Planar patch clamp electrodes |
-
2002
- 2002-05-21 FR FR0206185A patent/FR2839979B1/en not_active Expired - Fee Related
-
2003
- 2003-05-16 WO PCT/FR2003/001496 patent/WO2003097787A1/en active Application Filing
- 2003-05-16 US US10/515,805 patent/US7527965B2/en not_active Expired - Fee Related
- 2003-05-16 AU AU2003260552A patent/AU2003260552A1/en not_active Abandoned
- 2003-05-16 JP JP2004506446A patent/JP4410848B2/en not_active Expired - Fee Related
- 2003-05-16 EP EP03752829A patent/EP1507846A1/en not_active Withdrawn
Non-Patent Citations (1)
| Title |
|---|
| See references of WO03097787A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| US20050233441A1 (en) | 2005-10-20 |
| JP4410848B2 (en) | 2010-02-03 |
| WO2003097787A1 (en) | 2003-11-27 |
| FR2839979A1 (en) | 2003-11-28 |
| AU2003260552A1 (en) | 2003-12-02 |
| JP2005525812A (en) | 2005-09-02 |
| FR2839979B1 (en) | 2004-08-20 |
| US7527965B2 (en) | 2009-05-05 |
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