JP2004524046A - Cell and tissue culture device with temperature control - Google Patents

Abstract

The present invention relates to a cell and tissue culture device, wherein the device contains at least one culture well (18-I), each containing at least one flexible pouch (6,7; 27,29), one of which is provided. A first (2) and a second (25) reservoir capable of receiving at least the culture, a link coupled to the well and the pouch for circulating the culture from one reservoir to the other via the well; Means for the pouches of the first (2) and second (25) reservoirs, respectively, determined by the control module (50) and designed to control the culture circulation in the wells; Pressurizing means for applying a second external pressure sequence, maintaining the first selected temperature inside the wells and culturing out of the first and second reservoirs to feed the wells The comprises a temperature control means to be monitored (49,51-58) by a control module which is designed to set the second selected temperature.
[Selection diagram] Fig. 1

Description

FIELD OF THE INVENTION
[0001]
The present invention relates to the field of dynamically culturing cells and tissues using a moving culture or nutrient medium.
[0002]
More precisely, the invention relates to a method comprising: i) one or more culture wells defining a chamber for containing cells or tissues to be cultured; and ii) each containing at least one flexible bag, And first and second reservoirs, at least one of which is suitable for containing a culture; iii) to allow the culture to flow from one reservoir through the wells to another reservoir. Iv) the first and second reservoir bags are external to a first and / or second sequence respectively determined by one or more control modules; and iv) a link means coupled to the well and the bag. Means for culturing cells and tissue, comprising: pressurizing means for allowing pressure to be applied and serving to govern the flow of culture medium in said wells. On.
[Background Art]
[0003]
Apparatus of the type described in French patent application 00/00548 allows suitable flow conditions to be maintained throughout the culture. However, when the culture needs to be under temperature control, this type of equipment must be placed inside a suitable incubator, thereby reducing the biological risk, cost, handling and size of the replacement. And it becomes impossible to observe how the culture is progressing using a microscope while the incubation is taking place. In addition, this movement leads to temperature changes, which can have deleterious biological consequences.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0004]
It is an object of the present invention to provide an original solution for all or some of the disadvantages identified above.
[Means for Solving the Problems]
[0005]
For this purpose, the invention relates to a device of the kind described in the introduction, wherein a temperature control means under the control of a control module is provided, and a first selected temperature or a first temperature in the well is provided. To maintain the selected temperature cycle and / or to supply the wells with at least one of the first and second reservoirs with a second selected temperature or a second selected temperature. An apparatus is provided that operates to apply two selected temperature cycles.
[0006]
Thus, the temperature regulation in the device can be performed either exclusively at the well level or exclusively at the level of the culture supplied to the wells, or in fact, the exchange between the culture and the cells can be performed. It can be performed simultaneously at both the well level and the culture medium level so as to minimize the temperature distribution when performing.
[0007]
The first and second temperatures (or first and second temperature cycles) are selected as a function of the type of culture. Thus, if required for culture, they can be substantially identical or they can be different. The second temperature (or second cycle) can also be varied between one reservoir and another, if desired. It is also possible to change the temperature (or cycle) during the progress of the culture. To do this, the parameters for changing the temperature may be programmed, for example by being included in a program for determining the external pressure sequence provided by the control module. Such programming may be performed using an input interface or by moving a pre-defined program into a memory of the device coupled (or integrated) to the control module and then to the program (The memory may optionally be rewritable via the interface).
[0008]
In a first embodiment of the device according to the invention, said temperature regulating means comprises a heating fluid circuit, said circuit being formed in a well (possibly in the form of a flow channel formed at the periphery of the chamber, or formed in the wall of the well and having a second At least a first part integrated in the wall forming the space for allowing liquid flow coupled to one connecting means, or in the wall forming the first and second reservoirs, respectively. Second and third portions, which are arranged to allow the flow of heat-carrying liquid to flow therethrough (these being an inner shell forming said first and second reservoirs when assembled together) (Which may be a space formed between the outer shells) or a combination of the first, second and third parts. In this combination, preferably, the second part is arranged to supply a heat-carrying fluid (liquid or gas) to the first part, while the third part comprises the first part. Arranged to select the flowed heat carrier fluid. Thus, in order to supply and withdraw the heat-carrying fluid, it is particularly advantageous that the second and third parts of the fluid circuit include second and third connecting means, which are connected to the gap between the shells. Open to the inside and each adapted to be connected to the first connection means and to the (main) fourth part of the fluid circuit.
[0009]
In this first embodiment, the heating fluid circuit preferably comprises a pump connected to a main container containing a fraction of the heat-carrying fluid (liquid or gas) and the first, second and third pumps. It includes electric heater means (eg, a heater resistor) for heating the heat-carrying fluid before it is supplied to the section in a controlled manner.
[0010]
In a second embodiment of the device of the present invention, the temperature regulating means comprises a first electric heater element (e.g., positioned against the wall of the well) for providing at least a portion of the controlled heating of the well. Or a second electrical heater element (e.g., the reservoir) for providing at least a portion of the controlled heating of the first and second reservoirs. Or a combination of said first and second electric heater elements).
[0011]
Naturally, a third embodiment of the device according to the invention, wherein the temperature regulating means comprises both a fluid circuit (as in the first embodiment) and an electric heater element (as in the second embodiment). It is also possible to assume.
[0012]
The device of the present invention may include additional features, particularly the following features, either separately or in combination:
Each of said first and second reservoirs may comprise a top or bottom connected by a narrow intermediate portion and link means for communicating a plurality of bottom bags, said top and bottom further leaking Not including entrance. At the same time, the pressurizing means is controlled by a control module to supply pressurized fluid at high or low pressure, or indeed intermediate pressure, to each of the top and bottom reservoir portions via said leak-free inlet. It may also include a fluid pump for supplying pressurized fluid at high pressure via (primary) first and second portions of the pressurization circuit connected to top and bottom pressure regulating valves. Good. In such an environment, it is particularly advantageous that the first part of the pressurizing circuit comprises a sub-part immersed in the heat-carrying fluid (liquid or gas), which is A second portion of the pressure circuit is housed in the main container to supply heated pressurized fluid. This makes it possible to minimize temperature disturbances on the culture. Further, the pressurizing circuit is housed outside the main container in contact with the heat transfer fluid to supply a pressurized fluid of a selected humidity to a second portion of the pressurizing circuit, and It is also possible to provide an auxiliary container (water-marie) for containing the humidifying fluid, which is supplied with fluid under pressure by a sub-part of the first part of the first part. This is particularly important when the flexible bag is semi-permeable.
[0013]
At least two, preferably three or four wells arranged in series and interconnected via link means, the first well being connected to the first reservoir while the opposite of said first well The side well may be connected to a second reservoir.
[0014]
The temperature control means may include at least one temperature sensor for providing the control module with a measured value representative of the temperature inside or near the well.
[0015]
A cover may be provided to isolate the well from the outside and possibly the reservoir, and indeed the entire device.
[0016]
The present invention also relates to a facility for culturing cells and tissues, comprising at least two devices of the above type arranged in parallel and controlling all of their control units, or Provide a facility to share one control unit that performs the functions of
[0017]
The facility may include a main fluid circuit for supplying the wells and / or reservoirs of each device in parallel. In this case, a central temperature control means controlled by the main control unit, for maintaining a selected common first temperature or a selected common first temperature cycle in the wells of each device; and / or Or serve to supply cultures flowing from at least one of the first and second reservoirs of each device to the well at a selected common second temperature or selected common second temperature cycle. It is particularly advantageous to provide central temperature control means.
[0018]
In one variation, the main control unit controls the temperature control means of each device so that they are independent of each other and have a first selected temperature or a first selected temperature in the well of the corresponding device. Maintaining the temperature cycle and / or exiting at least one of the corresponding first and second reservoirs, independently of each other, at a second selected temperature or a second selected temperature cycle. In the well.
[0019]
The facility may also include a main cover that simultaneously isolates the wells of each device from the outside, possibly with associated reservoirs or complete devices.
BEST MODE FOR CARRYING OUT THE INVENTION
[0020]
Other features and advantages of the invention will be apparent from a review of the following detailed description and the accompanying drawings.
[0021]
The accompanying drawings are essentially defined in nature. Thus, they can contribute not only to explain the invention, but also to limit it where appropriate.
[0022]
First, reference is made to FIGS. 1-4 to describe the cell and tissue culture device in a non-limiting example.
[0023]
The device 1 shown in FIG. 1 first comprises a first reservoir 2 having a top 3 connected to a bottom 4 via an intermediate part 5. This reservoir 2 is formed by a rigid wall 15 which gives the reservoir a constant volume, which will be further described below.
[0024]
In the example shown, the top 3 of the reservoir contains a flexible top bag 6. Similarly, the bottom 4 contains a flexible bottom bag 7, which is connected to the top bag 6 via a duct 8 contained in the middle section 5 and the top of the first reservoir 2 and The bottoms 3 and 4 are housed close together so as to be isolated from each other.
[0025]
The top bag 6 has an inlet / outlet 9 adapted to cooperate in a leak-free manner with a top opening 10 formed in one of the top partitions of the first reservoir 2. Thus, the top bag 6 may be connected to a top access control means 11 which is itself coupled to a culture or gas supply module, or, as shown, preferably pressurized nutrients (or culture fluid). It may be connected to the container 14. For reasons of compactness, the nutrient container 14 is located below the top of the reservoir 2, but it could be located elsewhere.
[0026]
Similarly, as shown in FIG. 1, the bottom bag 7 has an inlet / portion adapted to cooperate with a leak-free opening formed in the wall or elsewhere of the bottom 4 of the first reservoir 2. It has an outlet 12, in which case it cooperates with an access control means 13 housed outside the bottom 4 of the reservoir 2.
[0027]
The bottom bag 7 may include two substantially rigid members to prevent it from flattening when subjected to very high pressure. Because it prevents good flow of the culture. Also preferably, in the intermediate section 5, the first reservoir 2 is provided with an additional opening allowing the liquid or gas to be manually or automatically injected into or withdrawn from the duct 8 Part. The opening is preferably fitted with a septum which is particularly suitable when the device for injection or withdrawal is a syringe fitted with a needle. It is also preferred to provide a partition at each of the bottom and top of the reservoir.
[0028]
Also, preferably, the top and bottom bags 6 and 7 are made of a porous material, at least from outside to inside. They may be made of silicone, polydimethylsiloxane (PDSM), actually polytetrafluoroethylene (PTFE), or actually dimethyl and methylvinylsiloxane polymers. This allows gas exchange between the culture fluid in the flexible bag and the gas trapped inside the top and bottom 3,4 of the first reservoir 2. These bags are made of different materials so as to provide different functions, especially with respect to the exchange of fluids contained within the reservoir (generally pressurized gas, which will be described in more detail below). In addition, bags in one reservoir give different shapes and volumes.
[0029]
In the example shown in FIG. 1, the bottom bag 7 communicates with the culture wells 18-1 to 18-3 via the access control means 13 and a bottom opening formed in the wall of the reservoir.
[0030]
The access control means 13 is preferably of the "pinch" type. They have a hollow end into which one end of the linking means 20 is inserted, which at its opposite end opens the first well 18-1 into the culture chamber 19-1. In the form of a duct (or tube) having This first culture chamber 19-1 is connected to the second culture chamber housed in the second well 18-2 via another link means 21, also implemented in the form of a duct (or tube). Communicates with 19-2. Similarly, the second culture chamber 19-2 is connected to the third culture chamber housed in the third well 18-3 via another link means 21 formed in the form of a duct (or tube). Is in communication with Finally, in this example, the last linking means 20 provides communication between the third culture chamber 19-3 and the second reservoir 25 described below.
[0031]
The second reservoir 25 is preferably substantially identical to the first reservoir 2 described above with reference to FIGS. Thus, in this example, the reservoir 25 includes a top 26 having a top flexible bag 27 contained therein, a bottom 28 having a bottom flexible bag 29 contained therein, and a top bag 27. And a narrow intermediate portion 23 for accommodating an intermediate duct 24 connecting the to the bottom bag 29. This duct 24 is likewise tightly housed in the middle part 23 so that the top 26 is airtightly isolated from the bottom 28.
[0032]
The top bag 27 has a suitable inlet / outlet 30 connected to an access control means 31 which, like the access control means 11, is connected to a gas or liquid supply 32 or an extractor. Can be connected. Similarly, the bottom bag 29 has an inlet / outlet 33, which in the example shown is connected to an access control means 34 located outside the bottom 28 in the second reservoir 25.
[0033]
The second reservoir 25 also preferably includes openings at its top, middle and bottoms 26, 23 and 28 so that liquid or gas can be manually or automatically introduced into the pocket or intermediate duct 24. Allowing it to be injected or withdrawn. These openings are preferably provided in each partition.
[0034]
In this example, the access control means 34 is also preferably of the “pinch” type and has a hollow end coupled to the end of the link duct 20 for this purpose.
[0035]
Therefore, between the top bag 6 of the first reservoir 2 and the top bag 27 of the second reservoir 25, via the culture chamber 19-i (i = 1 to 3 in this example) and the link members 20, 21. A circuit is formed.
[0036]
In order to be able to control the growth of the culture thermally, the device regulates the temperature inside the culture well, or regulates the temperature of the culture supplied to the well, or Preferably, a temperature control means as shown in FIGS. 1 to 4 is provided to control both the temperature in the well and the temperature of the culture solution.
[0037]
In the embodiment shown in these figures, the temperature of the culture is adjusted by circulating a heat-carrying fluid (liquid or gas) inside their rigid walls 15 in two reservoirs 2 and 25. More precisely, the wall 15 defining the top and bottom of the reservoirs 2 and 25 has a fluid circulation space 35 incorporated therein, which forms part of a fluid circuit for heating purposes I have. As shown in FIGS. 2 to 4, this circulation space 35 is advantageously formed by assembling the inner shell 16 and the outer shell 14 containing the inner shell 16 together.
[0038]
The inner shell 16 is preferably constructed by assembling together two shell halves 16a and 16b forming the inner portions of the top, middle and bottom of each reservoir 2 and 25.
[0039]
The outer shell 17 also preferably has a first retaining member 36 (in this case an orifice) cooperating with a second retaining means 37 (in this case a stud) formed on the outer surface of the inner shell 16. It is constructed by assembling two shell halves 17a and 17b together. At its top it also has an inlet 42 fitted in a first connector 43 (suitable for connection to the "external" main part of the heating fluid circuit), and at its bottom it has an end wall It has an outlet 44 with a second connector 45 suitable for connection to a third or fourth connector 46 adapted to 18-1 and 18-3. As a result, the heat-carrying fluid can be circulated inside the walls 15 of the reservoirs 2 and 25 to provide an effective temperature control for the culture circulating in the bag.
[0040]
A channel 47 is provided which forms another part of the heated fluid circuit to provide temperature regulation within the well. When the well 18 is made in a thick solid block 48, the channel 47 is preferably formed by creating a hollow in said block 48 at the periphery of, preferably below, the zone defining the well. Is done. In one variation, when the well and reservoir are installed on a support plate, the support plate may include a channel 47 for circulating a portion of the heat-carrying fluid beneath the well 18. . These channels 47 are provided on one side of a third connector 46 for connection of the first reservoir 2 to the second connector 45, and for connection of the second reservoir 25 to the first connector 43. Connected to the other side of the fourth connector.
[0041]
The heat carrier fluid circulates through the main part of the heating fluid circuit, reaches the well 15 of the first reservoir 2 via the first connector 43, circulates in the inter-shell space 35, and then To the connector 45. It then flows through the third connector 46 into the channel 47 of the well 18 and reaches the fourth connector 46. It then flows into the wells of the second reservoir 25 via its second connector 45, circulates through the inter-shell space, and then reaches the first connector 43, from which the heated fluid circuit Return to the main part of.
[0042]
To enable the heat transfer fluid to circulate, the main part of the fluid circuit includes a main container 49, which first contains a fraction of the heat transfer fluid, and an electric heater means 51, such as an adjustable heater. A heater resistor, an inlet 52 connected to the first connector 43 of the second reservoir via a duct 53, and a pump for feeding the first connector 43 of the first reservoir 2 via another duct 55 ( (Not shown). This further duct 55 is preferably provided with two parallel connected solenoid valves (or pneumatic valves) 56 and 57 and a pressure sensor 58 (or pressure contact) for regulation purposes. The temperature of the heat transfer fluid in the main container 49 is selected such that the culture at the outlet 12 of the bottom bag 7 contained in the first reservoir is at a temperature suitable for the purpose of culture.
[0043]
Of course, the temperature inside the well may be different or substantially the same as the temperature of the culture exiting the first reservoir, as required.
[0044]
In one variation, reservoirs 2 and 25 and well 18 may be supplied in equilibrium with the same heat carrier fluid, or may be supplied with heat carrier fluid coming from two or three independent heating fluid circuits. Good. It is also possible to provide a heated fluid circuit for each part of the reservoir. This makes it possible to provide a reservoir containing the culture medium, arranged at different temperatures on both sides of the culture chamber, so as to form a temperature profile or a temperature cycle.
[0045]
The source (or nutrient container) 14 and / or gas or liquid supply (or waste container) 32 may also be used to maintain their contents at a selected temperature, which may be arbitrarily different. They may have their own thermostat circuit. This thermostatically controlled temperature may include heating or cooling. Generally, it is preferred to maintain them at a temperature in the range of about 3 ° C to about 12 ° C to ensure that the media is stable.
[0046]
In another completely different variant, the temperature adjusting means comprises an electric heater means such as a heater resistance or a controlled temperature profile (CTP) element. Such means may be located at or at selected locations within the wall defining the reservoir and / or well.
[0047]
It is also possible to envisage combining heater resistance and heating fluid circuits.
[0048]
Additionally, one or more temperature sensors may be provided at selected locations to provide temperature measurements to the control unit to improve temperature control within the wells and / or reservoirs.
[0049]
In order to control the internal volumes of the top bags 6 and 27 and the bottom bags 7 and 29, the device of the present invention includes pressurizing means described below with reference to FIG.
[0050]
In the embodiment shown, a common pressurizing means is used for the two reservoirs 2 and 25, and these means, together with most of the temperature control means, are connected to an external unit 105 (for example, the unit represented by dashed lines in FIG. A). However, in one variant, each reservoir may have its own pressurizing means housed under its respective unit location, for example below the top of the reservoir.
[0051]
The pressurizing means comprises a pressure booster (or pump) 60 which is supplied with ambient air 61 and supplies a pressurized supply 62 which is preferably coupled to a pressure sensor (or pressure contact) 63. A circuit 59 is provided. Vessel 62 feeds main duct 64 with pressure regulator 65 and then feeds first flow regulator 66 and particle filter 67 (eg, having a 0.01 micron grid). When the device is used with a plurality of different pressurized fluids (e.g., air and carbon dioxide), an auxiliary duct 68 is provided that is supplied with an auxiliary fluid 69 (e.g., carbon dioxide), and the auxiliary duct is provided with a second pressure. It has a regulator 70 followed by a second flow regulator 71 and feeds the main duct 64 between the first pressure regulator 65 and the filter 67. In such an environment, it is advantageous to provide a third flow rate regulator 72 between the filter 67 and the point where the auxiliary duct 68 is connected.
[0052]
The main duct 64 serves to supply compressed fluid between the two reservoirs 2 and 25 and to the culture medium container 14. The pressurized fluid is heated by a heat carrier fluid located in the main container 49 to minimize temperature disturbances caused by the pressurized fluid penetrating the top and bottom of the reservoirs 2 and 25. To do this, a portion of the main duct 64 is housed in the main container 49, preferably in a coil or any form that facilitates heat exchange.
[0053]
In addition, an auxiliary container 74 is preferably provided in the main container 49 and partially filled with humidified liquid so that the humidity of the pressurized fluid can be controlled before penetrating into the reservoirs 2 and 25. The main duct portion 73 which is immersed in the heat-carrying fluid is open into the auxiliary container.
[0054]
The part 75 of the main duct 65, which opens into the auxiliary container 74, is preferably connected via a thermometer / hygrometer 76 to four valves 78, 79, 80 and 81 connected in parallel. Provided at a high pressure (eg, about 45 mbar) to a first port 77 provided, and secondly at a high pressure (eg, about 45 mbar) to a second port 82 opened into the culture medium container 14. Thirdly, four valves 84, 85, 86 and 87 connected in parallel are connected to a third port 83 provided with a fourth flow rate regulator 88 preferably located upstream of these valves. A fourth port 89 that supplies at low pressure (eg, about 10 mbar) and has a fourth, preferably fifth, flow regulator 90 followed by a solenoid valve 91 (or pneumatic valve) and a pressurized supply 92. At a moderate pressure (e.g., about 30 mbar), and this pressurized source 92 is connected to the four De valve or air valve) to supply.
[0055]
In one variation, different pressurized fluid circuits may be provided to control the volume of the bag contained within the top and bottom of the same reservoir. This makes it possible to use different pressurized fluids in the same reservoir so that the bags perform different functions, for example to perform a comparative test.
[0056]
The various solenoid valves (or air valves) 78-81 and 84-87 are preferably all three-way valves (two inlets and one outlet), and the solenoid valves (or air valves) 78-81 are solenoid valves (or Or pneumatic valves) 84-87, respectively, each of which acts via connectors 93-96 which are connected to connectors 39, 41 mounted on leak-free inlets 38, 40 respectively. The interior of the top and bottom 3,4 of the first reservoir 2 and the top and bottom 26,28 of the second reservoir 25 determine the volume of the flexible bag contained therein.
[0057]
Also, these solenoid valves (or pneumatic valves) are preferably of the "pinch" type as described above, for example access control means for wells 18 and flexible bags, as described in patent application FR 00/00548 Can also be used to determine the state of However, this is only one possible example, and a switch or valve could be used.
[0058]
All solenoid valves and pressurized fluid pumps are controlled by an electronic control unit 50, which for this purpose is preferably connected to a link interface 97 (eg RS232 type) so that it can be remotely controlled by a process computer. A connected microprocessor (or microcontroller) mounted on the card is provided.
[0059]
When programmed, the microcontroller 50 will operate the solenoid valve (or, if necessary) to apply a high and / or low pressure sequence to the bag with pressurized fluid at the top and bottom 3,4 of the reservoirs 2 and 25. Control the air valve). Of course, the microcontroller 50 may include a memory 98 containing a plurality of culture programs, preferably a rewritable memory, wherein each culture program has a respective volume of the various flexible bags, and wells and / or wells. Alternatively, the first and second pressure sequences are determined to determine the regulated temperature of the heat transfer fluid.
[0060]
As mentioned above, instead of using a microcontroller dominating one pressurization circuit, for example, the same microcontroller dominates two at least partially independent pressurization circuits located below the top of these reservoirs. It is possible to use a controller. In another variation, it is possible to use two independent microcontrollers that are pre-synchronized to dominate two independent pressurization circuits.
[0061]
The device preferably includes a cover for isolating one or more wells from the external medium and possibly isolating the reservoir from the external medium. This serves not only to avoid exchanges occurring through the various diaphragms, but also to limit temperature disturbances. This also serves to establish a "mechanical" protective barrier around the well. Also, this cover can cover the entire device, thereby forming an enclosure defining a biological barrier, which is particularly useful when the device itself is not placed under a laminar flow hood. The shape and material of the cover can be selected so that the cells and tissues contained within the wells can be viewed under a microscope or using other suitable optical means while culturing them. To this end, the cover is preferably made of a material that is not breakable and is transparent on the wells.
[0062]
Also, outlets for connection to atmospheric pressure with solenoid valves (or air valves) 99-102 under the control of the control unit may be provided at the top and bottom of the reservoirs 2, 25. In addition, as shown in FIGS. 2-4, the inlets 9, 30 of the top flexible bags 6, 27 are preferably in rigid ducts 103 defined by the rigid walls of the inner shell halves 16a, 16b. And a respective top cavity provided with a discharge means (not shown) so as to remove any microbubbles of air which may be formed in the flexible bags of the reservoirs 2 and 25 during operation. 104 are provided.
[0063]
The device of the present invention can be viewed as lacking a control unit coupled to a consumable member (reservoir and / or well), perhaps for a single use only. This simply provides an external control, a pressurization and temperature control unit 105 with first and second connection means 93-96, 106 each connected to a pressurization and temperature control circuit, and secondly Providing two reservoirs 2 and 25 of each device, each having a top and bottom within a portion of the reservoir, and third and fourth connection means 39, 41 and 43 connected to the intershell sace 35; This can then be achieved by connecting the first connecting means 93-96 to the third connecting means 39, 41 and connecting the second connecting means 106 to the fourth connecting means 43.
[0064]
To start a new culture, the used consumables (reservoirs and / or wells) are removed, replaced with new consumables and connected to an external control unit.
[0065]
As schematically shown in FIGS. 5 and 6, a plurality of devices 1 are arranged in parallel to culture cells and tissues for high throughput (same culture) or high differentiation (different cultures). It is possible to configure the equipment. In this example, the equipment has four parallel devices 1-1 to 1-4, and each device 1-i (in this case, i = 1 to 4) has three cultures connected in series. It has wells 18-j (in this case, j = 1 to 3). The reservoirs with their respective heat-carrying fluid circuit spaces are interconnected, for example by mounting the studs 37 on the inner shell half 16b through suitable holes 108 formed in the outer shell half 17a, 17b ( See FIG. 6).
[0066]
These devices can be completely independent of each other. In such an environment, they may have a common control unit that controls the pressurization and temperature regulation circuits that are independent of each other, or independent control units that each control one pressurization circuit and one temperature regulation circuit. You may have. In such an environment, the adjustment of temperature and / or pressurized fluid may be different for each device. However, such devices may be interdependent as some of those wells may be in communication.
[0067]
It is also possible to envisage equipment in which the devices have wells independent of one another by sharing a common pressurization circuit and a common temperature regulation circuit controlled by a common (or main) control unit. . In such an environment, the main parts of the pressurizing means and the temperature adjusting means and the main control unit are housed in an external unit 105 (represented by a broken line in FIG. 1). As a result, it is possible to configure equipment in which the device constitutes a "consumable" type modular part, possibly for one-time use. This simply provides an external control, pressurization and temperature control unit 105 connecting the first connection means 93-96, 106 to the pressurization circuit and the temperature control circuit, respectively, second, third and third Fourth connection means 39, 41 and 43 are provided respectively for the two reservoirs 2 and 25 of each device connected to the top and bottom inside the reservoir and to the intershell space 35, respectively, and then said first connection means 93 to 96 Is connected to the third connection means 39, 41, and the second connection means 106 is connected to the fourth connection means 43.
[0068]
To proceed with a new culture, the used consumables (containers and / or wells) are removed and replaced with new consumables in which the wells are optionally inoculated with cells.
[0069]
In such equipment, the number of devices connected in parallel can be varied as needed.
[0070]
In the installation of the invention, as in the device of the invention, the culture wells 18-j are connected in series on a support plate 107 as shown in FIG. 5, or they hollow a thick solid block 48. May be formed directly (as shown in FIG. 1).
[0071]
In the first example (FIG. 5), the support plate 107 divides each of the wells 18i-j (in this case i = 1-4, j = 1-3) and a fraction of the heat-carrying fluid into these wells. A housing can be provided for receiving a channel 47 for circulation in close proximity to the environment. The support plate 107 may also have channels or ducts for circulating a fraction of the pressurized fluid. In this second embodiment, the culture wells of the device can be made in independent solid blocks or in one block. Details regarding embodiments of wells suitable for use in the device of the present invention are given in patent application FR 00/00548.
[0072]
As described above when describing the device 1, the main cover is to be isolated from the outside and, if possible, to isolate the two reservoirs in each device of the installation, or in fact all of the device, if possible. It is advantageous to provide This can be avoided by using a cover for each device.
[0073]
An example of implementing the device and equipment of the present invention (i.e., the first and second series of pressures for determining the volume of the reservoir bag) can be found in patent application document FR 00/00548. It is merely recalled here that the installation and device are suitable for operation in the various modes described below.
[0074]
The "laminar flow" mode raises the culture into one of the top reservoirs of the two reservoirs, and then establishes the culture so as to establish a height difference between the top and bottom bags of the two reservoirs. Flows under gravity from the middle reservoir to the bottom reservoir and is present when raising the culture to the top bag of another reservoir. This same operation is then repeated in the opposite direction ("return direction") to perform one complete cycle (retaliation) through the well between the two reservoirs. The number of cycles is a function of the type of culture performed in well 18. The four steps of the retaliation cycle in laminar flow mode are grouped together in FIG. The number of consecutive cycles is chosen as a function of the type of culture to be performed.
[0075]
The “turbulent” mode exists when the high pressure is continuously applied to the bottom bags 7 and 29 of the first and second reservoirs 2 and 25. That is, the first and second sequences of the top bags of the first and second reservoirs are constituted by a sequence of four low pressure cycles. This mode has only two steps that are grouped together in the form of the "round trip" cycle of FIG. The number of consecutive cycles is selected as a function of the type of culture performed. This mode may be implemented in the first variant (FIG. 9), in which high pressure is not continuously maintained in the two bottom bags 7 and 29 and high pressure is applied to the top bags 6 and 27. Maintained continuously. This allows the culture to flow quickly between the two bottom bags 7 and 29, even if the liquid cannot rise anymore due to the high pressure in the top bags 6 and 27. In a second variant (not shown), the first sequence applied to each bag of the first reservoir comprises a first high pressure cycle with a second low pressure cycle alternately, The second sequence applied to each bag in the reservoir consists of a low pressure first cycle with an alternately high pressure second cycle.
[0076]
The two modes of operation described above, namely laminar and turbulent modes, and deformation modes, are merely a few of the many examples possible. Therefore, it is possible to combine a turbulent operation cycle with a laminar operation cycle.
[0077]
The present invention applies to a great variety of cells and tissues, particularly as listed below:
-Intestinal cells: intestine 407, Caco-2, Colo 205, T84, SW 1116, WiDr, HT 29, HT 115, HT 55;
-Endothelial cells: human aortic smooth muscle cells (HAOSMC);
Epithelial cells: human neonatal epithelial keratinocytes (NHEK-Neopooled), horse dermis;
・ Cancer cells: HeLa, CHO-K1;
Intestinal fibroblasts: CCD-18Co;
· MRC-5, 3T3, Wi-38 type fibroblasts;
・ Myeloma: SP20-Ag14, P3X63 Ag8 653, MPC11;
・ Hybridoma;
-Insect cells: SF9.
[0078]
This list is not exhaustive in any way, but merely provides an example.
[0079]
The invention is not limited to the modes of operation of the devices and equipment described above merely by way of example, but rather, on the contrary, all modifications that will occur to those skilled in the art within the scope of the appended claims. It covers examples.
[0080]
Thus, in the foregoing, a temperature regulation circuit has been described in which the heat transfer fluid is circulated around the temperature rise. However, for freezing certain media, for example, preserves, an auxiliary temperature control circuit may be used in which the circulating heat carrier fluid acts to remove heat. Of course, in such an environment, the device of the present invention needs to be adapted to the cooling medium under the control of the control module.
[0081]
Further, in the above description, the wells are placed at a first selected temperature and / or the fluid is placed at one or more second selected temperatures. However, it is also possible to envisage placing the wells under a first temperature cycle or profile and / or placing the fluid under a second temperature cycle or profile.
[0082]
In addition, the inlet sections of each reservoir and chamber can be adjusted to independently control their respective temperatures, especially when provided by a common heat transfer circuit.
[0083]
Finally, the temperature regulating means may be arranged to provide a thermal shock to the chamber and / or well. This is particularly advantageous when it is necessary to modify the state of the cell membrane. The heat shock may be combined with a pressure change achieved by controlling the fluid flow rate and / or the internal pressure of the chamber.
[Brief description of the drawings]
[0084]
FIG. 1 is a fragmentary schematic cross-sectional view of a culture apparatus of the present invention having a plurality of chambers.
FIG. 2A is a perspective view of the two inner shell halves of the reservoir of FIG. 1 before assembly together.
FIG. 2B is a perspective view of the two inner shell halves of the reservoir of FIG. 1 after assembly together.
FIG. 3 is a perspective view of the two inner shell halves of FIG. 2 prior to assembly with the two outer shell forms of the reservoir;
FIG. 4A is a perspective view, prior to assembly, showing how the inner shell halves are positioned inside the corresponding outer shell halves.
FIG. 4B is a perspective view after assembly showing how the inner shell halves are positioned inside the corresponding outer shell halves.
FIG. 5 is a schematic perspective view of a culture facility constituted by four culture devices arranged in parallel.
FIG. 6 is a perspective view showing an assembly of four double shell reservoirs for a facility of the type shown in FIG. 5;
FIG. 7 is a diagram showing a series of eight successive reciprocation steps for a culture solution in a laminar flow mode.
FIG. 8 is a diagram showing a series of four successive reciprocation steps for a culture solution in a turbulent flow mode.
FIG. 9 shows a modification of the turbulent flow mode shown in FIG.

Claims (30)

An apparatus for culturing cells and tissues, the apparatus comprising:
At least one culture well (18-i) suitable for containing cells or tissues to be cultured;
A first and a second reservoir, each containing at least one flexible bag (6, 7; 27, 29), at least one of which is suitable for containing a culture medium; 2, 25);
Link means (20, 21) coupled to the well and the bag to allow the culture to flow from one reservoir through the well to the other;
A first and / or second sequence defined by at least one control module (50), respectively, for flowing the culture through the wells relative to the first and second reservoirs (2, 25); Pressurizing means arranged to apply an external pressure of
Maintaining a first selected temperature or first selected temperature cycle inside the well (18-i) and / or flowing out of the first and second reservoirs to supply the well Apparatus characterized by including temperature control means (35, 47, 49, 51-58) controlled by said control module to bring the culture to a second selected temperature. Apparatus according to claim 1, wherein the temperature regulating means is integrated into a wall defining the well (18-i) and wherein a heat carrying fluid is configured to circulate therein. A device comprising a fluid circuit, including a portion (47). Apparatus according to claim 2, wherein the first part of the fluid circuit opens into a circulation channel (47) for a heat-carrying fluid incorporated in the wall of the well (18-1). And a first connection means (46). Apparatus according to any one of the preceding claims, wherein the temperature control means is integrated in a wall (15) defining a first and a second reservoir (2, 25), respectively. And a fluid circuit comprising a third portion (35) and configured to allow heat transfer fluid to circulate therethrough. 5. Apparatus according to claim 4, wherein each of said first and second reservoirs assemble together an inner shell (16) and an outer shell (17) containing said inner shell, and heat transport therein. A device characterized by being created by defining an inter-shell space (35) through which fluid can circulate. Apparatus according to claim 2 or 3 in combination with claim 4 or 5, wherein the second part (35) of the fluid circuit is adapted to supply a heat-carrying fluid to the first part (47). An apparatus, wherein the third portion of the fluid circuit (35) is configured to recover heat transport fluid circulated through the first portion (47). Apparatus according to the combination of claims 3, 5 and 6, wherein the second and third parts (35) of the fluid circuit open into the inter-shell space (35). (43, 45), and a first sub-portion is connected to the first connection means, and a second sub-portion is connected to a fourth portion of the fluid circuit for supplying and recovering a heat transfer fluid. An apparatus characterized by being suitable for being connected. Apparatus according to any of claims 2 to 7, wherein the fluid circuit comprises a pump coupled to a main container (49) containing a fraction of the heat transfer fluid; And electric heater means (51) for heating the heat-carrying fluid in a controlled manner before being supplied to the third part (47, 43 and 45). 9. Apparatus according to any one of the preceding claims, wherein the temperature adjusting means provides at least some of the controlled heating of the wells. An apparatus comprising: Apparatus according to any one of the preceding claims, wherein the temperature regulating means comprises a second electrical power supply for providing at least a portion of the controlled heating of the first and second reservoirs. An apparatus comprising a heater element. Apparatus according to claim 9 or 10, wherein the electric heater element comprises a heater resistor fixed to a wall defining a reservoir and / or a well. Device according to any one of the preceding claims, wherein the first and second reservoirs (2, 25) are interconnected via a narrow intermediate portion (5, 23). 3, 26) and a bottom (4, 28), each top and bottom of the first and second reservoirs containing a respective bag, and the top and bottom flexible bags (6, 27). 7, 29) communicate with each other via said intermediate portion (5, 23), and said link means (20, 21) communicate with said bottom bag (6, 29); The top and bottom (3,26; 4,28) of the second reservoir (2,25) each further comprise a leak-free inlet (38.40), and said pressurizing means comprises: High pressure pressurized fluid is passed through a first portion of the pressurization circuit (64) to top and bottom valves (78, 81) controlled by a control module (50). , 84, 87; 79, 80, 85, 86) connected to a second portion of the pressurizing circuit (77, 82, 83, 89), and the first and second Suitable for supplying pressurized fluid at high, low or medium pressure via said leak-free inlet (38, 40) to each of the top and bottom (3, 26; 4, 28) of the reservoir An apparatus comprising a fluid pump (60). Apparatus according to the combination of claims 8 to 12, wherein a first part of the pressure circuit (64, 65) is heated to the pressure circuit (77, 82, 83, 89). An apparatus comprising a sub-portion (73) immersed in a heat-carrying fluid contained in said main container (49) to provide a fluid. 14. Apparatus according to claim 13, wherein said pressurizing means comprises an auxiliary container (74) arranged in said main container (49) in contact with said heat carrying fluid, said container comprising a humidifying fluid. And pressurized fluid supplied by a sub-portion (74) of a first portion of the pressurized circuit to be supplied to a second portion of the pressurized circuit (65). Device characterized by exhibiting variable humidity. Apparatus according to any one of the preceding claims, comprising at least two wells (18-i) arranged in series and communicating with each other via the linking means (21). An apparatus wherein one of the wells (18-1) is connected to the first reservoir (2) and the other well (18-3) is connected to the second reservoir (25). . 16. The device according to claim 15, wherein a third well (18-2) is arranged in series between the two wells and communicates with each of the wells via the linking means (23). ). Apparatus according to any of the preceding claims, wherein the control module (50) comprises: the pressure sequence and the first and second selected temperatures or the first and second selected temperatures. A device comprising a rewritable type memory (98) suitable for storing a selected temperature cycle. Apparatus according to any one of the preceding claims, wherein the control module (50) connects the respective reservoir and the inlet section of the chamber when they are supplied by a common heat transport circuit. , Devices arranged to independently control their temperatures. Apparatus according to any one of the preceding claims, comprising at least one nutrient container (14) and a gas or liquid supply (32) connected to a thermostat circuit, and the control module. (50) The device characterized in that the contents of each of the nutrient container (14) and / or the gas supply device (32) are arranged to maintain a selected temperature. 20. Apparatus according to any one of the preceding claims, wherein each reservoir portion is located at a different temperature in the reservoir portions located on opposite sides of the chamber, such that a temperature cycle or temperature is established in the well. An apparatus, wherein the apparatus is connected to a heated fluid circuit so that a profile can be formed. 21. Apparatus according to any one of the preceding claims, wherein the temperature adjusting means is arranged to apply a temperature shock to the interior of the chamber and / or well. 22. Apparatus according to any one of the preceding claims, wherein the temperature adjusting means comprises at least one temperature sensor adapted to apply a measured value representing the temperature in a well to the control module. An apparatus characterized in that it comprises: 23. Apparatus according to any one of the preceding claims, comprising a cover for isolating the wells from the outside at least. 24. The device of claim 23, wherein the cover is for isolating the well and the reservoir therapy from the outside. 25. A facility for culturing cells and tissues, comprising: a main control unit for controlling at least two devices (1-i) according to any one of claims 1 to 24 together with said devices. Equipment characterized by being provided with 50). 26. The facility according to claim 25, comprising a main fluid circuit that supplies the wells and / or reservoirs of each device in parallel. 26. An installation according to claim 25, controlled by the main control unit (50) and maintaining the same selected first temperature or the same selected first temperature cycle inside the wells of each device. And / or supplying the culture fluid flowing out of at least one of the first and second reservoirs of each device to the wells at the same second selected temperature or the same selected second temperature. An installation comprising central temperature control means arranged to be subjected to a second temperature cycle. 28. The facility according to claim 27, wherein the main control unit controls the temperature control means of each of the devices so that they independently of each other select a first temperature or a second temperature within a well of the device. Try to maintain one selected temperature cycle and / or they work independently of each other to flow out of at least one of the first and second reservoirs of the device to be supplied to the wells A facility for subjecting the culture to a second selected temperature or a second selected temperature cycle. The facility according to any one of claims 25 to 28, further comprising a main cover for isolating at least the well of each apparatus from the outside. 30. The facility of claim 29, wherein the main cover serves to simultaneously isolate the well and the reservoir of each device from the outside.

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