Culture medium dispensing system for automated cell culture systems.

Abstract

Device (10) for distributing liquid in a T-flask type cell culture chamber characterized in that it comprises: - a stopper (20) suitable for sealing a T-flask type culture chamber (30), - a first channel (40) extending from the outside to the inside of said stopper (20) and associated with said stopper (20), characterized in that said first channel (40) extends substantially parallel to the longitudinal axis of said stopper (20) and in that the internal end of said first channel (40) opens into a distribution head (44), and a T-flask type culture chamber (30) associated with said stopper (20).

Description

Title of the invention: Culture medium distribution system for cell culture automation. Technical field of the invention

[0001] The present invention belongs to the field of scientific equipment. More particularly, the present invention belongs to the field of fluid circulation control. The device according to the invention is more particularly intended for the distribution of culture medium to T-Flask type cell culture dishes. State of the art

[0002] Cell culture is an essential element of biological research. It is used in particular for the study of cellular behavior and the production of recombinant proteins or viruses. For this purpose, adherent or suspended cells are cultivated in a liquid medium inside gas- and temperature-regulated incubators.

[0003] Outside of industrial production devices, cell culture requires a lot of human manipulation. The cells are grown in chambers (commonly called culture dishes) placed in a dedicated incubator. To feed the cells and remove the waste produced, an operator must regularly renew the culture medium by removing used medium and reintroducing fresh medium. In addition, cell density must be controlled. Too low or too high a density prevents their homogeneous growth. As a result, the operator must regularly remove cells in excess quantity to discard them or distribute them to other culture dishes. All these manipulations consume time (including weekends and holidays) and materials (e.g. pipettes).Furthermore, the regular opening and closing of culture boxes and bottles containing the culture medium are significant sources of bacterial contamination.

[0004] There is therefore a significant need to eliminate human intervention as much as possible in the process of in vitro cell culture.

[0005] Automated devices are already commercially available. However, they cost several hundred thousand euros to install and require many dedicated consumables. These devices are suitable for industrial production but not for the scale of a research and development laboratory.

[0006] Ideally, a cell culture machine should be able to adapt to the equipment already existing within the laboratory. More specifically, a cell culture machine should be able to adapt to commercially available culture dishes.

[0007] There are two main types of culture chambers depending on the type of cells. cultivated. First, there are culture chambers dedicated to the culture of non-adherent cells. These dishes often take the form of cylindrical bottles. There are also dishes dedicated to the culture of adherent cells. The latter have a parallelepiped part comprising a flat internal face on which the cells will adhere, grow and divide. The parallelepiped part is extended by a prism, forming the neck, then by a threaded neck receiving the cap of said dish. The longitudinal axis of this threaded neck forms an angle with respect to the plane of the culture surface. These are also known as T-Flask.

[0008] The manual filling and renewal of the T-Flask with culture medium is carried out by placing the dishes vertically. In this position, the culture medium is deposited at the bottom of the dish (i.e. on the face perpendicular to the culture face). When the dish is repositioned horizontally, the culture medium is distributed evenly over the culture surface and covers all the cells.

[0009] When using T-Flask in an automated cell culture system, it is not desirable to have to handle or shake the dishes to evenly distribute the culture medium on the surface of the cells. This is particularly relevant for the culture of certain cell types such as neurons which are particularly sensitive to shaking.

[0010] However, in the absence of manipulation or agitation, the culture medium can be distributed randomly, in particular due to surface tension, inside the dish, forming heterogeneous zones at which the cells will die.

[0011] In cases of partial renewal of medium, there is also a need for the new medium not to concentrate in particular areas of the dish and cause heterogeneous growth of the cells.

[0012] There is therefore a need for a device allowing the uniform distribution of culture medium inside T-Flask without agitation of the latter. Summary of the invention

[0013] The present invention relates to a device for distributing liquid in a T-flask type cell culture chamber, characterized in that it comprises: - a cap suitable for sealing a T-Flask type culture chamber, - a first pipe extending from the outside to the inside of said plug and associated with said plug, characterized in that said first pipe extends substantially parallel to the longitudinal axis of said plug and in that the internal end of said first pipe opens into a distribution head.

[0014] In the context of the present invention, the term “T-Flask” refers to a cell culture enclosure, comprising a polygonal portion, preferably substantially parallelepipedal, comprising a flat cell culture surface for the culture of adherent cells.

[0015] In the context of the present invention, the term "plug" refers to a removable part congruent with the opening of said culture enclosure and capable of closing all or part of said opening of said enclosure.

[0016] In the context of the present invention, the term “associate” is intended to mean that said pipe and said plug are integrally linked.

[0017] In the context of the present invention, the term "internal" is intended to designate the end of the pipe intended to be arranged inside the T-Flask.

[0018] According to a preferred embodiment, said dispensing head comprises at least one spray nozzle.

[0019] In the context of the present invention, the term “sprinkler nozzle” refers to a part connected on the one hand to said first pipe and comprising an opening communicating towards the outside.

[0020] According to a preferred embodiment of the invention, said dispensing head comprises several spray nozzles distributed over at least two planes.

[0021] According to a preferred embodiment of the invention, said device further comprises a T-Flask type culture chamber associated with said cap.

[0022] According to a preferred embodiment of the invention, said distribution head is arranged directly above the cultivation surface.

[0023] According to a preferred embodiment of the invention, said dispensing head is directly above the proximal end of the culture surface of said T-Flask.

[0024] In the context of the present invention, the term "proximal" refers to the position of an element relative to said plug. The element closest to the plug is said to be "proximal". In contrast, the element furthest away is said to be "distal".

[0025] According to a preferred embodiment of the invention, said device further comprises a second pipe, intended to extract the liquid from said T-flask.

[0026] According to a preferred embodiment of the invention, the second pipe extends substantially parallel to the longitudinal axis of said plug and is associated with said plug and opens into a suction head.

[0027] According to a preferred embodiment of the invention, said suction head is arranged at the level of the culture surface. Even more preferably, said suction head is arranged at the intersection of the culture surface and the side wall of the T-Flask.

[0028] According to a preferred embodiment of the invention, said device further comprises a pump arranged in such a way that it is capable of circulating liquid in said pipeline towards said distribution head.

[0029] According to a preferred embodiment of the invention, said pump is a variable flow pump.

[0030] The present invention also relates to a method for distributing a culture medium in a T-Flask type cell culture chamber using a device according to the invention, characterized in that it comprises a step consisting of circulating, via said pump, culture medium in said first pipe by varying, continuously or discontinuously, the working flow rate of said pump between two previously defined flow rates. Brief description of the figures

[0031] [Fig.l] is a top view of an embodiment of a device according to the invention.

[0032] [Fig.2] is a front view of the same embodiment of a device according to the invention.

[0033] [Fig. 3] is a left view of the same embodiment of a device according to the invention.

[0034] [Fig.4] is a front view of the same embodiment of a device according to the invention in which the T-flask has been removed.

[0035] [Fig.5] is a top view of the same embodiment of a device according to the invention in which the T-flask has been removed.

[0036] [Fig.6] is a right-hand view of the same embodiment of a device according to the invention in which the T-flask has been removed. Detailed description of the invention

[0037] With reference to [Fig.l]-[Fig.6] the device 10 according to the invention comprises a stopper 20 closing the opening of a T-flask 30.

[0038] T-Flask 30s are well known to the cell culture specialist. Various models can be found sold under this name by companies such as Sigma-Aldrich®, Thermo-Fisher scientific® or Ciba-Coming®. They are generally sold with a cap that can be screwed onto their neck 31. The device 10 according to the invention will replace this cap and allow both the T-Flask to be closed and the passage of the fluids necessary for cell culture.

[0039] The cap 20 can be indifferently associated with the T-Flask 30 by screwing onto the outside of the neck 31 and / or by friction inside the neck 31.

[0040] Preferably, the cap 20 is associated by screwing to the neck 31. Natively, the neck 31 of a T-flask is provided with a thread 32. Consequently, the cap 20 preferably comprises a thread 21 congruent with the thread 32.

[0041] The plug 20 preferably fits into a cylinder whose longitudinal axis merges with the axis of rotation of the stopper 20 around the neck 31. The stopper 20 preferably has a lateral external wall 24, delimiting an internal volume 25 intended to receive the neck 31. The stopper 20 also has at one of its ends an external wall called a closing wall 26 closing the internal volume 25, and at its other end an opening 27 allowing the passage of the neck 31.

[0042] Advantageously, the closure wall 26 comprises a filter allowing the passage of gases between the inside and the outside of the T-Flask.

[0043] According to another embodiment of the invention not shown, the stopper 20 may comprise a flexible cylindrical body intended to be inserted inside the neck 31 and to completely close it.

[0044] The stopper 20 is preferably made of plastic and even more preferably of polypropylene.

[0045] The stopper 20 can in particular be manufactured by molding or by additive synthesis.

[0046] The external surface of the cap 21 may advantageously comprise gripping means 22, 23 facilitating its handling and / or its placement on the neck 31. Said gripping means 22, 23 may in particular take the form of one or more portions of hexagonal external section arranged on the external surface of said cap 20. According to a preferred embodiment of the invention, the cap 20 comprises a portion of hexagonal external section 22 near the opening 27 and a portion of hexagonal external section 23 near the closure wall 26.

[0047] The stopper 20 comprises one and advantageously at least two pipes 40, 50. The first pipe 40 is intended to bring the culture medium inside the T-Flask 30. The pipe 50 is intended to take the culture medium from inside the T-Flask 30.

[0048] The first pipe 40 extends from the outside to the inside of the plug 20. It preferably comprises from its external end to its internal end a connection means 41 intended to allow the connection of the pipe 40 to a flexible tube, and a portion 42 extending inside the plug 20.

[0049] The connection means 41 preferably extends from the external face of the closure wall 26 and even more preferably perpendicularly thereto. The connection means 41 is preferably made up of a rigid tube comprising at its end an external bulge 43. The latter has an external diameter greater than the internal diameter at rest of the flexible tube intended to be associated with said connection means 43. In doing so, the external bulge 43 will allow association by friction when the connection means 43 is inserted into said flexible tube.

[0050] The portion 42 communicates with the connection means 43. It is preferably rigid and rectilinear and extends from the inside of the closure wall and even more preferably perpendicular to the latter.

[0051] The portion 42 preferably has a diameter between 1.5 and 3 mm.

[0052] Preferably, the portion 42 extends beyond the opening 27 of the plug 20 so that its end is positioned, under normal conditions of use, directly above the proximal end of the culture surface.

[0053] According to a preferred embodiment of the invention, the portion 42 opens into a distribution head 44 comprising one or more spray nozzles 45.

[0054] The dispensing head 44 communicates on the one hand with the portion 42 and opens on the other hand, towards the outside of the dispensing head 44, via the spray nozzles 45.

[0055] The role of the distribution head 44 is to split, when necessary, the flow coming from the portion 42 into several flows opening into the spray heads 45.

[0056] To do this, the distribution head 44 preferably comprises a hollow chamber communicating on the one hand with the portion 42 and on the other hand with the spray heads 45. Preferably, said hollow chamber has a volume of between 300 and 900 mm3, even more preferably between 450 and 750 mm3 and most preferably between 550 and 600 mm3.

[0057] The dispensing head 44 preferably has the shape of a disc or a parallelepiped. In the case of a disc, even more preferably, said disc has a flow inlet arranged on its edge and a series of at least one spray nozzle on the portion of this same edge opposite said flow inlet. In the case of a parallelepiped, said parallelepiped preferably has a flow inlet arranged on one of its faces and a series of at least one spray nozzle on the opposite face.

[0058] The spray nozzles 45 are oriented so as to be able, depending on the feed rate, to send the culture medium to a predefined area of ​​the culture surface. The present invention may comprise several spray nozzles 45 each having a different orientation to allow a homogeneous distribution of the culture medium over the entire culture surface.

[0059] Preferably, said spray nozzles 45 are a pipe open to the outside. Even more preferably, they have a circular cross-section and a diameter of between 0.5 and 1 mm.

[0060] In the context of the present invention, "orientation" is intended to designate the angle that, under normal conditions of use, the portion of the pipe directly upstream of the external opening of the spray nozzle 45 makes relative to the horizontal and the vertical. Advantageously, this latter portion has a length of between 3 and 5 mm.

[0061] Other distributions of the spray nozzles 45 are conceivable. In particular, the spray nozzles 45 can be distributed into several series, each of at least two nozzles sprinkler nozzles 45, each of the series being at different heights. The sprinkler nozzles 45 of two different series may be placed in a staggered pattern or aligned with the adjacent nozzle directly above or below.

[0062] Three preferred embodiments of a sprinkler head according to the invention are presented below.

[0063] According to a first embodiment, the spray head comprises six spray nozzles 45 oriented so as to form an angle of between 18 and 22° and preferably an angle of 20° with the horizontal. Furthermore, said spray nozzles 45 are oriented relative to the longitudinal plane of symmetry of said device so as to form an angle of 17.1°, 10.5° and 3.5°. The model being symmetrical, the angles are therefore identical on the other side relative to said plane.

[0064] According to a second embodiment, the spray head comprises six spray nozzles 45. The six openings are arranged in two superimposed rows of three openings. The orifices of the upper row make it possible to reach the bottom of the bottle when the flow rate is maximum, the spray nozzles 45 of the upper row are oriented so as to form an angle of 20° with the horizontal. The central spray nozzle 45 of this row follows a trajectory in the azimuthal plane. The two lateral nozzles 45 are arranged so as to form an angle of 17.3° on either side of this plane.

[0065] The spray nozzles 45 of the lower row make it possible to reach the middle of the bottle. They form an angle of -!❖ with the horizontal. The jet coming from the central hole of this row follows a trajectory in the azimuthal plane. The central spray nozzle 45 of this row follows a trajectory in the azimuthal plane. The two lateral nozzles 45 are arranged so as to form an angle of 29.4° on either side of this plane.

[0066] According to a third embodiment, the sprinkler head comprises six sprinkler nozzles 45 arranged on a single horizontal plane. The two central nozzles are oriented so as to form an angle of between 18 and 22° and preferably an angle of 20° with the horizontal. The next two nozzles are oriented so as to form an angle of 9° with the horizontal. The two external nozzles are oriented so as to form an angle of -6° with the horizontal. Furthermore, said sprinkler nozzles 45 are oriented relative to the longitudinal plane of symmetry of said device so as to form an angle of 30.2°, 12.6° and 9.7°. The model being symmetrical, the angles are therefore identical on the other side relative to said plane.

[0067] According to a preferred embodiment of the invention, said device further comprises a second pipe, intended to extract the liquid from said T-flask.

[0068] The second pipe 50 extends from the outside to the inside of the plug 20. It preferably comprises from its external end to its internal end a connection means 51 intended to allow the connection of the second pipe 50 to a flexible tube, and a portion 52 extending inside the plug 20.

[0069] The connection means 51 preferably extends from the external face of the closure wall 26 and even more preferably perpendicularly thereto. The connection means 51 is preferably made up of a rigid tube comprising at its end an external bulge 53. The latter has an external diameter greater than the internal diameter at rest of the flexible tube intended to be associated with said connection means 53. In doing so, the external bulge 53 will allow association by friction when the connection means 53 is inserted into said flexible tube.

[0070] The portion 52 communicates with the connection means 53. It is either rigid or flexible and extends from the inside of the closure wall towards the inside of the enclosure of the T-Flask.

[0071] Preferably, the portion 52 extends beyond the opening 27 of the plug 20 so that its end is positioned, under normal conditions of use, directly above the proximal end of the culture surface.

[0072] According to a preferred embodiment of the invention, the portion 52 opens into a suction head 54 whose distal end is arranged in contact with the culture surface and even more preferably at the intersection of the culture surface and one of the side walls of the culture enclosure. In doing so, said suction head 54 will be able to collect all of the used culture medium present in the culture enclosure.

[0073] Advantageously, the suction head 54 is a rigid pipe communicating with a flexible portion 52. The connection between the suction head 54 and the portion 52 are advantageously arranged in a sliding manner in a fixed guide cylinder 55.

[0074] In this latter embodiment, by sliding in the guide cylinder 55, the suction head 54 will be able to adapt to all sizes of T-Flask. Thus, according to a preferred embodiment of the invention, said suction head has a length of between 50 and 100 mm and even more preferably a length of between 60 and 80 mm.

[0075] The suction head 54 communicates on the one hand with the portion 52 and opens on the other hand, towards the outside of the closure head 54 at its end 55.

[0076] The first pipe 40 and / or the second pipe 50 are advantageously connected to at least one pump allowing the supply and removal of the culture medium.

[0077] According to a preferred embodiment of the invention, said pump is a variable flow pump and even more preferably a peristaltic pump.

[0078] According to a preferred embodiment of the invention, said pump has a working flow rate of between 10 and 300 mL / min and even more preferably between 250 and 300 mL / min.

[0079] It is thus understood that under normal conditions of use, the cap supplied with the T-flask will be replaced by a device according to the invention. In doing so, the first pipe 40 and the second pipe 50 will extend inside the neck. The portions 42, 52 of each of the pipes 40, 50 will preferably stop at the intersection of the neck 31 and the culture enclosure 30.

[0080] To supply the enclosure with culture medium, the pump will supply the first pipe 40 for a first predefined duration with a first predefined flow rate. In doing so, the culture medium will form a jet at the outlet of the spray nozzles 45 and reach the culture surface at specific points. During this first step, one or more “puddles” of culture medium will form on the culture surface.

[0081] By continuously or discontinuously varying the flow rate of the pump, a displacement of the point of impact of the jet of culture medium on the culture surface will be obtained and thus an equal distribution of the culture medium over the entire culture surface will be possible.

[0082] The pump connected to the first pipe 40 and the pump connected to the second pipe 50 may be identical or different.

[0083] In order to remove the culture medium from the culture enclosure, the pump connected to the second pipe 50 is set in motion. The suction head 54 being located at the level of the culture surface, it allows total or partial removal of the culture medium present in the enclosure 30.

[0084] According to a preferred embodiment of the invention, the pump is controlled by a programmed and / or programmable computer device so as to cause the pump to perform different supply / withdrawal cycles in culture medium.

Claims

Claims

1. Device (10) for distributing liquid in a T-flask type cell culture chamber characterized in that it comprises: - a plug (20) capable of closing a T-Flask type culture chamber (30), - a first pipe (40) extending from the outside to the inside of said plug (20) and associated with said plug (20), characterized in that said first pipe (40) extends substantially parallel to the longitudinal axis of said plug (20) and in that the internal end of said first pipe (40) opens into a distribution head (44), and a T-Flask type culture chamber (30) associated with said plug (20).

2. Device (10) according to the preceding claim characterized in that said dispensing head (44) comprises at least one spray nozzle (45).

3. Device (10) according to one of the preceding claims 1 to 2, characterized in that said distribution head (44) comprises several spray nozzles (45) distributed on at least two planes.

4. Device (10) according to one of the preceding claims, characterized in that said distribution head (44) is arranged directly above the cultivation surface.

5. Device (10) according to the preceding claim, characterized in that said dispensing head (44) is directly above the proximal end of the culture surface of said T-Flask (30).

6. Device (10) according to one of the preceding claims, characterized in that said device (10) further comprises a second pipe (50), intended to extract the liquid from said T-Flask (30).

7. Device (10) according to the preceding claim characterized in that the second pipe (50) extends substantially parallel to the longitudinal axis of said plug (20) and is associated with said plug (20) and opens into a suction head (54).

8. Device (10) according to the preceding claim, characterized in that said suction head (54) is arranged at the level of the culture surface.

9. Device (10) according to one of the preceding claims, characterized in that said device (10) further comprises a pump arranged in such that it is capable of circulating liquid in said pipe (40) towards said distribution head (44).

10. Device (10) according to the preceding claim characterized in that said pump is a variable flow pump.

11. Method for distributing a culture medium in a T-Flask type cell culture chamber (30) using a device (10) according to claim 10 characterized in that it comprises a step consisting of circulating, via said pump, culture medium in said pipe (40) by varying, continuously or discontinuously, the working flow rate of said pump between two previously defined flow rates.

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