FR2849862A1 - Cell culture device for performing in vitro assays, useful e.g. for evaluating pharmaceutical or cosmetic agents, comprises a removeable chamber containing a support and system for controlling operational parameters - Google Patents

Abstract

Cell culture device (1) for performing at least one in vitro assay that includes a removeable chamber. Cell culture device (1) for performing at least one in vitro assay comprises: (1) at least one chamber (2), which holds at least one support (17); (2) at least one device for controlling at least one operating parameter of (2), during use; and (3) at least one system for supplying fluid to, and removing it from, (2). The operating parameters are temperature, pressure, humidity and optionally also at least one other parameter of a medium containing the cells on (17). The new feature is that the chamber is removeable. An Independent claim is also included for an in vitro test method that uses the new device.

Description

DEVICE AND METHOD FOR CULTURING CELLS FOR IN VITRO TESTING

  The invention relates to a device mainly intended for the cultivation of cells, for example in the form of tissue, for carrying out tests in vitro.

  Today there are various devices for performing in vitro cultures. In addition to increasing the number of simultaneous cultures, it is also sought to reduce the sources of measurement error, the handling time during a culture and to improve the automation of the measurements.

  Thus there are devices for carrying out in vitro cultures which include the use of at least one chamber, comprising at least one support means such as for example a well microplate as described in patent application FR 01 / 11.923 of the plaintiff. Such devices make it possible to improve the automation of the measurements, mainly in terms of controlling the temperature, the pressure, and other possible parameters - such as the pH - of the generally liquid or gel medium comprising the cells, and any fluid admitted to said chamber. The fluid admitted into the chamber generally comprises at least one gas chosen from the group formed by carbon dioxide, air, water vapor, oxygen and nitrogen. However, the improvement may not be as effective in practice as the theory allows, 2 2849862 mainly for regulations other than thermoregulation, and more particularly for the study of cellular functions of cells placed in different contexts. experimental, such as natural state, stress, pollution, UV, thermal radiation, before and / or after treatment of said cells with active principles molecules or finished products for pharmaceutical, cosmetic or environmental health purposes, etc. ; or simply the capacity for resistance, tolerance or recovery of cells following stress. By "stress" is meant an attack of a physicochemical nature, or more generally a reaction involved in all relations between the organism and the environment which surrounds it. There are generally different types of stress: radiation, irradiation with at least one radiation, oxidation, thermal, chemical, gas, heavy metals, arsenite, physiological (by at least one element chosen from the group formed by hormones, growth factors, cell cycle, and amino acid analogs), pathophysiological (infections, hypoglycemia, anoxia); typically the three types of stress most commonly studied on cells are, separately, gas stresses, radiation type stresses and thermal type stresses. In addition, such devices generally do not allow the use of at least one fluid comprising at least one polluting gas, for example chosen from the group formed by exhaust gases, ozone, and cigarette fumes. In fact, the presence of residues left by the polluting gas 30 implies a high risk of degradation by contamination of the measurement (s) over time. Finally, no device known to date makes it possible to carry out the three types of stress described above simultaneously, in the case where the gas is a gaseous pollutant.

  There are also other cellular incubation devices, such as those marketed by the companies JOUAN or KENDRO, of very large capacity (typically 150 liters, with several hundred microplates), which is difficult to manage and more precisely to homogenize in particular at the temperature. Furthermore, such devices are mainly used for mass cell culture.

  In addition, if the majority of the devices of the prior art generally allow and under certain conditions to carry out a relatively satisfactory cell culture, they do not, for the reasons mentioned above, make it possible to carry out a study of the 20 cellular functions and in particular the most adequate cellular stress possible, mainly in terms of reproducibility and multiplicity of stress.

  This is all the more so since the fluids circulated at least temporarily in the chamber comprise at least 25 a polluting gas. Furthermore, such devices require very large quantities of daily gas, in particular for the condition of hypoxia of the cellular material.

  4 2849862 The device according to the invention overcomes the problems described above.

  Thus, the device according to the invention is a cell culture device for carrying out at least one in vitro test, said device comprising at least one chamber in which there is at least one support means, and said device comprising at least one means for regulating at least one operating parameter of said chamber during the in vitro test use of said device, said parameter being chosen from the group formed by temperature, pressure, hygrometry and possibly at least one other possible parameter of a possible medium comprising the cell supported by said support means, said device further comprising at least one means for entering the chamber of at least one fluid and at least one means for leaving the chamber at least one fluid, said device being characterized in that said chamber is removable.

  Advantageously, such a device according to the invention allows the study of the cellular functions of cells placed in different experimental contexts, such as natural state, stress, pollution, UV, thermal radiation, before and / or after treatment of said cells by principles. active molecules or finished products for pharmaceutical, cosmetic or environmental health, etc. ; as well as the capacity for resistance, tolerance or recovery of cells 2849862 following stress. Said device advantageously makes it possible to study simultaneously the three most commonly studied types of stress, which are gas stresses, radiation type stresses (by irradiation) and thermal type stresses. The device according to the invention can also be qualified as a stress generator, that is to say as a stress conditioner.

  By "at least one cell" is meant according to the invention any unicellular microorganism, such as a virus, a bacterium or a parasite, as well as any multicellular organism, such as a cellular tissue of animal or vegetable origin. By "test" according to the invention is meant the conditioning, for example in temperature and / or in pressure and / or in hygrometry, optionally in the presence of a fluid, as well as the generation of stress in vitro, which generation may be preceded, continued and / or analyzed outside the device according to the invention, for example by placing said cell in a conventional incubator or in a device for measuring at least one given parameter.

  The device according to the invention is advantageously and most often an apparatus for conditioning cellular stress. Thus, said device is therefore preparation equipment, mainly for non-analytical purposes. The impact of stress is generally assessed by existing techniques: cytotoxicity tests (LDH, Glucose, XTT, MTT, RN, PAC, PAL, GSH, CVDE, SRB ...), genotoxicity (AO / BET, comet, micronuclei), molecular 6 2849862 (quantitative PCR, cDNA bio chips ...), proteomics (ELISA, protein bio chips ... etc.

  The support means according to the invention is generally chosen from the group formed by microplates with wells (6, 12, 24, 48, 96, 384 or 1536 wells), such as for example that described in patent application FR 01 / 11.923 by the applicant, the test tubes, the micro test tubes, the Petri dishes, the flasks or the culture dishes for example of 12.5, 25, 75 and 125 cm 2, possibly themselves supported by at least one tray-type support.

  According to the invention, the possible medium comprising the cell or cells and supported by said support means is generally, and most often, under the conditions of use, in liquid or gel form, and the cell or cells are immersed in said middle. Said medium can be monitored (that is to say measured in its structure and / or function and / or nature) and optionally and preferably regulated by at least one parameter, for example chosen from the group formed by the pH, the bicarbonate concentration and carbon dioxide level. Preferably, the pH is regulated to a value of 6.9 to 7.4, the concentration of bicarbonate is regulated to approximately 24 mM in the culture medium, and the level of carbon dioxide is regulated to approximately 5% by volume. in the total gas phase, as is known to those skilled in the art. Said medium, called culture medium, generally comprises at least one element chosen from the group formed by inorganic salts, amino acids, vitamins, cofactors, nucleosides, ribonucleosides and glucose.

  According to the invention, the culture medium can also comprise at least one pollutant, generally liquid and / or solid, supplied for example by pipette before and / or after the use of the device according to the invention. Such a pollutant comprises, for example, a molecular active ingredient against which the reaction of the cells is to be tested, or else a finished product for health and / or cosmetic and / or environmental use, such as, for example, an insecticide or a pesticide, of which we want to test the safety and / or efficacy on the cells. In this case, said pollutant is well supplied before using the device according to the invention. However, such a pollutant may also contain a molecular active agent for which the reaction of the stressed cells is to be tested, or else a finished product for health and / or cosmetic and / or environmental use the safety of which is to be tested and / or the efficiency on stressed cells. In this case, said pollutant is well supplied after the use of the device according to the invention. Those skilled in the art obviously adapt any use of the device according to the invention according to their needs and knowledge.

  According to the invention, the term "fluid" means any liquid and / or gas or any other intermediate state, which may optionally contain a few solid particles (for example cigarette smoke comprises 8 2849862 in particular tar in the form of solid particles) According to the invention is understood to mean by "chamber" any closed, preferably sealed, means in which at least one cell is disposed on which at least one in vitro test can be carried out. Thus, and preferably, the chamber of the device according to the invention is sealed and hermetically closed. The device according to the invention therefore generally and preferably comprises at least one means for sealing said chamber from the environment outside said chamber. By "removable" is meant according to the invention which can be moved (e) at least temporarily from a position qualified as initial and replaced (e) at least temporarily substantially in its initial position, and this generally at will. The fluid which can be admitted into the chamber generally comprises at least one gas chosen from the group formed by carbon dioxide, air, water vapor, oxygen and nitrogen. In a particularly advantageous manner according to the invention, the fluid which can be admitted into the chamber generally comprises at least one polluting gas, for example chosen from the group formed by exhaust gases, cigarette fumes and ozone.

  More generally, such a polluting gas can be chosen from the group formed by motor vehicle exhaust gases such as nitric oxide and carbon monoxide; cigarette smoke such as nicotine, tar, carbon monoxide, tar droplets, polonium, nickel, hydrocarbons, acrolein, nitro derivatives, aromatic amines, hydrocyanic acid 9 '2849862 , silic acid, carbonic acid, acetic acid, formic acid, benzoque acid, phenols, acetone, acetaldehydes, formaldehyde, toluene, ethanal, ammonia, lead, methanol, hydrogen sulfite, vinyl chloride, cadmium, arsenic, mercury, formalin, pesticides, insecticides, titanium dioxide, phosphorus, naphthalene, methyl chloride, acetonitrile, nitrous oxide, and pyridine; asphyxiating gases; corrosive gases; 10 toxic gases by inhalation such as hydrogen sulfide, sulfur dioxide, sulfur trioxide, phosphine, bromine, hydrogen bromide, hydrogen fluoride, and petroleum gas; very toxic gases as qualified by regulation; rare gases as defined by the nomenclature of periodic elements; and ozone. Such a list is of course given as an indication and the person skilled in the art can easily, in the light of his knowledge, complete it. This advantageously allows the study of gas stress by the device according to the invention.

  The temperature in the chamber when the device according to the invention is used is generally from approximately 20C to approximately 50C, preferably from ambient temperature (of the external medium, typically approximately 200C) to approximately 500C, even more so. preferred from about 270C to about 45'C. The temperature is thus, as is known to those skilled in the art, generally about 370C for human cells 2849862, 340C for human sex cells, 270C for insect cells, and 220C for cells of fish. The pressure in the chamber when using the device according to the invention is generally less than or equal to 4 bars, or 0.4 MPa.

  The humidity in the chamber when using the device according to the invention is generally in the range of 50 to 99.999% water (in relative humidity), preferably 90 to 99.99% water ( in relative humidity 10). The pH of the medium, if any, when using the device according to the invention is generally in the range from 6 to 8, preferably from 6.8 to 7.4.

  The environment external to the device during its use complies with the usual rules of use, in terms of laboratory equipment and industrial equipment, as is known to those skilled in the art.

  According to the invention, the temperature in the chamber is generally and preferably regulated by at least one temperature regulation means comprising at least one means for thermal insulation of the chamber from the external environment and at least one means for measuring the temperature, associated with at least one adjustment means, which is for example at least one heating and / or cooling means. The temperature measurement means is for example and typically a temperature probe. Traditionally, said insulation means is the heating and / or cooling means, for example in the form of a heating enclosure comprising water which can be heated by at least one heating means. suitable heating, for example consisting of at least one heating resistor present in water, and which can be cooled (and / or 'heating) by at least one suitable means, for example consisting of at least one means external to said water circulation enclosure capable of supplying water to said enclosure at a desired temperature. The temperature measuring means is for example and typically a temperature probe. Advantageously, the device according to the invention therefore allows the study of thermal stress.

  According to the invention, the pressure in the chamber can be regulated by at least one pressure regulating means. For example the variation of gas mixture can allow, to a certain extent, to regulate the pressure in the chamber.

  According to the invention, the oxygen content in the chamber is generally regulated by at least one oxygen regulating means comprising at least one means for measuring the oxygen level, typically an oxygen probe, associated with at least one adjustment means, typically at least one means for entering oxygen or nitrogen. 25 For oxygen regulation, it is advisable according to the device of the invention to have at least three fluid inlets, namely, air, carbon dioxide and oxygen or nitrogen, combined by at least one mixing means which regulates the conditions for admitting the mixture into the chamber. Thus, for the implementation of hyperoxia condition (case of an oxygen level generally greater than about 21% by volume), the mixing means which is a gas mixer is associated with at least one bottle of air (bringing about 21% by volume), at least one bottle of carbon dioxide (generally to ensure a carbon dioxide level of 5% by volume) and at least one bottle of oxygen to provide the amount of additional oxygen desired. 10 For the implementation of the hypoxia condition (oxygen level generally less than approximately 21%), the gas mixer is associated with at least one air bottle (providing the approximately 21% by volume), at least a bottle of carbon dioxide (to ensure a level of carbon dioxide of 5% by volume) and at least one bottle of nitrogen to provide an additional quantity of nitrogen which will cause the oxygen level to decrease relatively, the whole being advantageously regulated by means of at least one means for measuring the oxygen level, typically an oxygen probe.

  In a particularly advantageous manner, the chamber of the device according to the invention is preferably of limited volume, for example from 3 to 6 liters. However, any volume of the chamber from approximately one milliliter to approximately ten liters can be envisaged according to the invention. This advantageously allows a setting in conditions, for example a setting in temperature or a setting in conditions of hypoxia or hyperoxia, relatively rapid and requiring a small amount of gas unlike conventional incubators.

  Advantageously, the chamber of such a device is disassembled relatively easily and, in particular, it can be cleaned and / or disinfected or even depolluted, for example and preferably by passage through an autoclave at high temperature and high pressure as well. that it is known to those skilled in the art, so as to remove, practically completely (see below) any possible source of contamination and / or any possible trace of product in said chamber. Said device is therefore easily reusable many times under excellent purity conditions. This is one of the many advantages of the invention, which allows excellent reproducibility of the in vitro tests carried out in said device.

  In a preferred embodiment, said device comprises at least two chambers, preferably from two to five, or even a plurality (ie at least three), typically in the case of use of polluting gas, of chambers according to the invention accommodating the cells, of which only one chamber can be used at a time within the device according to the invention. It is possible in the context of the invention to have said chambers in a conventional large capacity incubator during the culture periods before and after the stress generated by the device according to the invention. The removable chamber of the device according to the invention makes it possible to maintain a temperature chain for example of around 370C during the transfer of the microplates possibly contained in said chamber from the conventional incubator to the device according to the invention. This avoids a thermal shock 5 and advantageously makes it possible to gain in security and in reproducibility. Thus, the device according to the intention advantageously operates in addition to at least one conventional incubator. Such a conventional incubator typically allows, as explained in description 10 of the prior art, a thermal and gaseous configuration (at least one gas chosen from the group formed by air, water vapor, l oxygen, nitrogen and carbon dioxide).

  The presence of at least two chambers advantageously makes it possible to increase the yield of the device according to the invention. In fact, during the use or cleaning and / or disinfection or even decontamination of a room, the device can operate with another room. In addition, this makes it particularly advantageous according to the invention to practically completely avoid any cross-contamination, since a given chamber is associated with a given type of fluid, for example comprising a polluting gas, or for a type of experiment. given. This is particularly advantageous in the case of the presence of microorganisms such as the prion, or heat-resistant bacteria, or of polluting gas (s) such as for example ozone, cigarette smoke or exhaust gases, such as 'explained above, because despite autoclaving at high temperature and high pressure, these compounds are extremely difficult to remove practically completely.

  The device according to the invention can also comprise at least one additional means for cleaning and / or disinfection and / or decontamination in situ, in particular in the case where at least one means for entering the fluid must be used for another fluid in later use. According to a variant, it is possible that at least part of said input means is disposable and interchangeable, and that another part of said means is cleaned and / or disinfected and / or decontaminated at the same time as the chamber.

  In a preferred embodiment, the device according to the invention can also comprise at least one fluid inlet means, per fluid capable of being used in the device according to the invention. Such an inlet means generally and preferably comprises at least one means of regulation, for example in the form of a needle valve (or any other known gas valve device), of the inlet flow rate of said fluid.

  In an embodiment, independent or not of the previous embodiment, the device according to the invention can also comprise at least one fluid outlet means, per fluid capable of being used in the device according to the invention.

  16 2849862 Preferably, the device according to the invention further comprises at least one additional means for cleaning and / or disinfection and / or decontamination in situ by fluid capable of being used in the device according to the invention, for the means of entry and / or exit of said fluid. Alternatively, it is possible that at least part of said input and / or output means is disposable and interchangeable, and that another part of said means is cleaned and / or disinfected and / or decontaminated at the same time as the bedroom.

  According to the invention, it is possible and preferred that the various input means possibly present in the device according to the invention are combined together in a mixing means itself combined with a single input means so that that only one mixing input means is connected to the chamber. Such a mixture inlet means generally and preferably comprises at least one regulating means, for example in the form of a solenoid valve (or any other known device of an all-or-nothing tap or electronically controlled flowmeter), of the inlet flow of said mixture.

  Such a regulation means is advantageously such that it generally allows said mixture to enter the chamber 25 in the form of injection and non-injection cycles, the number, flow rate and duration of which can be programmed beforehand.

  According to the invention, the different outlet means 30 possibly present in the device according to the invention are generally and preferably combined together in a mixing means itself combined with only a mixing outlet means so that that only one mixing outlet means be connected to the chamber. Optionally, and in particular in the case of pressure regulation in the chamber, said outlet means can be regulated by at least one regulating means such as a valve or a solenoid valve as described above. On the other hand, it is possible according to the invention to recover the outlet fluid from the device according to the invention and then to inject it into a second device according to the invention; this can for example be the case when evaluating the activity of a depolluting bacteria.

  In a particularly preferred embodiment of the device of the invention, only one outlet means is present and directly connected to the chamber.

  According to the invention, the fluid outlet means or means are associated with at least one recovery means and / or at least one means of evacuation towards the outside.

  Of course, such means must comply with all the standards, in particular health, in force. According to the invention, any fluid inlet or outlet means, including the mixture inlet means mentioned above, of the device according to the invention generally and preferably comprises at least one filter means. so as to reduce and if possible avoid any possible contamination of the chamber by the fluid during the use of said device. For example, such a filtration means is a filtration membrane.

  The device according to the invention also comprises at least one means for supplying said chamber with water vapor. The water which is used is preferably deionized or distilled water. For example, said device generally comprises at least one means for storing water, for example located within the chamber, so as to maintain a sufficient degree of humidity within said chamber when the device is used according to the invention. This is particularly advantageous in the case of the use of cell culture medium, since this medium generally tends to evaporate over time by evaporation of the water which it can contain. Thus, the humidity is generally almost stable within the chamber during the use of the device, which practically completely prevents the drying of the possible culture medium. This virtual absence of drying therefore advantageously preserves its osmolarity (that is to say the inorganic salt concentration of said medium, which is typically 280 to 300 mOsm / kg for an animal cell culture medium).

  In view of the importance that this water content can assume, the device according to the invention generally comprises at least one means for measuring the water content in said chamber, for example a humidity probe. According to one embodiment of the device according to the invention, said device further comprises at least one means of irradiating at least a part of the chamber with photons and UV, individually or simultaneously. Said means of irradiation can be a means of access to irradiation by external light, as will be explained below. However, it can also include at least one additional means of irradiation external to the chamber, which is optionally provided in addition to the device according to the invention. Such an additional means generally irradiates at least part of the light spectrum such as visible, UV, IR, and is for example chosen from the group formed by UV, IR, visible and laser light sources.

  For example, for irradiation with external light, said chamber is of substantially parallelepiped shape and comprises various removable drawers each comprising at least one support means which is for example a microplate with wells, for example at 6, 12, 24, 96, 384, 1536 or 3072 wells, preferably 96 wells, said drawers being superimposed on each other.

  Then, according to this example, the upper part of the chamber comprises a cover typically in the form of a removable plate which allows, in the closed position, to maintain the darkness inside the enclosure, said cover covering a permeable window. (in glass for visible light or, interchangeable, in quartz for UV) with photons (possible use of high pass filter, low pass 30, band pass, block pass or even a 20 'mirror 2849862 dichroque to obtain a spectrum or a specific wavelength) and UV (UVa and / or UVb and / or UVc), typically in the form of a plate, which constitutes said irradiation means. In this case, the upper drawer 5 then generally constitutes said part of the chamber which is capable of being irradiated. Of course, such a window is generally equipped with at least one sealed insulation means, for example such a window is surrounded by at least one silicone-type seal. Even more preferably, said window is equipped with at least one heating means, preferably self-regulating at the same temperature as the chamber. Such a heating means advantageously makes it possible to avoid, when using the device according to the invention, condensation on the face 15 of the window pane inside the chamber.

  The invention also relates to a method of carrying out at least one in vitro test, in which at least one cell is placed on at least one support means, said support means being placed within at least one chamber, said cell being subjected to at least one parameter chosen from the group formed by temperature, pressure, hygrometry and possibly at least one other possible parameter of a possible medium comprising the cell, and to at least one fluid generally comprising at least one gas chosen from the group formed by carbon dioxide, air, water vapor, oxygen and nitrogen, and at least one polluting gas chosen as described above.

  In a preferred embodiment, said fluid comprises at least one polluting gas chosen as described above.

  Preferably, said method is such that, before and / or after carrying out the test, the cell is subjected to at least one parameter chosen from the group formed by temperature, pressure, hygrometry and possibly at least one other possible parameter of a possible medium comprising the cell, and at least one fluid generally comprising at least one gas chosen from the group formed by carbon dioxide, air, water vapor, oxygen and l 'nitrogen.

  In one embodiment of the method according to the invention, said method comprises any parameter analysis such that, before and / or after carrying out the test, the cell is subjected to at least one analysis of at least one parameter.

  In a particularly preferred manner according to the invention, said method comprises the use of the device according to the invention.

  The invention also relates to the method as described above comprising at least one cell culture. Such a culture generally lasts from 24 to 96 hours. In particular and preferably, such a method comprises at least one study of cellular stress. 30 Cellular stress is generally carried out over a short period of 0 to 180 minutes, but may, depending on the needs of the experimenter, take longer, over almost 96 hours for example. By cell is meant which applies to at least one cell, the cells generally and most often being in the form of a single-cell or multi-cell organism. In a particularly preferred manner according to the invention, the method according to the invention comprises at least one cell culture and / or at least one cell stress study in the presence of at least one polluting gas, generally chosen from the group formed by exhaust gases, ozone, and cigarette fumes, and / or at least a curative or preventive study of a stress of liquid and / or solid health and / or cosmetological pollutant for pharmaceutical and / or cosmetological purposes .

  Indeed a stress study can allow the evaluation of the tolerance of the resistance and the recovery of an organism.

  Such a method according to the invention comprises, in an almost reproducible manner, all other things being equal, at least one of the following operations, on at least one cell: * Evaluation of the efficacy of anti-cancer products * Study in radiotherapy * Evaluation of photo toxicity and photo protection of cosmetic product * Evaluation of dermocosmetic product against pollution 23 '2849862 * Evaluation of tolerance and / or resistance and / or recovery to a polluting gas and / or to stress cell * Assessment of genotoxicity, carcinogenesis or mutagenesis of gaseous, liquid or solid origin * Assessment of cytotoxicity of gas or even gas mixtures and polluting gas (es) * Assessment of toxicity and / or effectiveness of insecticide and / or pesticide * Thermal shock study (typically by changing from a set temperature value - for example 370C - to a higher temperature value , stable and homogeneous, for example 42 or 450C) * Study of hypoxia, hyperoxia or anoxia 15 * Study of aging * Study of ischemia and / or reperfusion (typically in the case of severe brain and / or heart damage) , * Fundamental study.

  Of course, all the analyzes linked to the studies mentioned above are carried out using known tests.

  The invention will be better understood and other characteristics and advantages will appear on reading

  the description which will follow, given for non

  limiting, with reference to Figures 1 to 5.

  FIG. 1 is a diagrammatic perspective view of a device according to the invention comprising a parallelepipedal chamber with three superimposed drawers.

  FIG. 2 schematically represents in perspective said chamber of the device according to the invention of FIG. 1.

  Figure 3 shows schematically in perspective said chamber of Figure 2, one of the drawers being open.

  Figure 4 shows schematically in perspective said chamber of Figure 2, one of the drawers 15 being open and its bottom (tray) being removed.

  Figure 5 shows schematically in perspective said chamber of Figure 2, the top of the chamber being removed.

  Identical numbers on different figures indicate similar objects.

  FIG. 1 schematically shows in perspective a device 1 according to the invention comprising a chamber 2 with three drawers 3, 4 and 5 superimposed, the drawer 3 being located in the upper position, the drawer 4 being located in the intermediate position and the drawer 5 being located in the lower position. A cover 6 covers the chamber 2, this cover 6 being removable. The cover 6 allows 2849862 to make it dark in the room, it can be folded down on one face of the device 1 by pivoting along hinges represented by the axis XX '. Chamber 2 can be removed by sliding upwards (slides not shown) once the cover 6 is folded down.

  The device 1 comprises a first part comprising said chamber 2, the cover 6 and an enclosure 10 constituted by a water jacket 10 allowing a heat exchange 10 on three faces of the chamber 2. The chamber 2 can for example be produced for the bottom and the sides in smooth-walled copper, thus promoting heat exchange with the water jacket 10. The front and rear faces of the chamber 2 are for example made of polypropylene. Chamber 2 can have a probe support on the rear side (not shown), for example for three oxygen, temperature and humidity probes, fixed for example by screwing on the bottom of chamber 2. The upper part of chamber 2 is a heating glass 20 18, which will be explained in FIG. 5. It comprises a glazed part 18b surrounded by a seal 18a, for example made of silicone. The window 18 is in fact formed by two superimposed plates between which there is a heating resistor, in the form of a microscopic film, self-regulated at the same temperature as (the atmosphere of) the chamber.

  The water jacket 10 includes a filling hole 10a and a drain hole 10b. 30 26 2849862 Each respective drawer 3, 4 and 5 of the chamber 2 is made for example of stainless steel and propylene. Sealing is ensured for each drawer 3, 4 and 5, by the use of two locking latches 5 which can be two screws respectively 3a and 3b, 4a and 4b, 5a and 5b, and a seal silicone, respectively 3d, 4d and 5d. A handle, respectively 3c, 4c and 5c, facilitates the opening of the respective drawer 3, 4 and 5. Each drawer 3, 4 and 5 has a removable tray, for example made of stainless steel. Only a tray 17 of the drawer 4 is shown in Figure 4 detached from the support 16 which constitutes the base of the drawer 4 and which fits into two slides (not shown) of the chamber 2. The tray 17 can support any means of 15 support as defined above; by extension it is considered that the plate 17 is part of said support means.

  The device 1 comprises a second part 20 consisting of a fluid compartment 7 comprising a gas mixer 9. A gas passage indicator 11 is located in this fluid compartment 7. In addition, three gas level indicators 9a, 9b and 9c are present, which can be regulated by the respective needle valves 8a, 8b and 8c. For example, in the case of use with a polluting gas, everything that is indexed by "a" relates to air, everything that is indexed by "b" relates to carbon dioxide and everything that is indexed by "c" corresponds to the polluting gas.

  27 2849862 The device 1 comprises a third part 7 consisting of an electrical compartment 12 comprising a control part 13 comprising a keyboard 13a and a control screen 13b.

  FIG. 2 schematically shows in perspective said chamber 2 of the device 1 according to the invention of FIG. 1. In addition to the three drawers 3, 4 and 5, each in the closed position, there is a device for locking the window heater 18, consisting of a rigid metal frame 15, for example made of stainless steel, and four bolts 14a, 14b, 14c and 14d which allow the frame 15 to be tightened on the glass 18 and thus ensure the sealing of the upper face of room 2.

  Figure 3 shows schematically in perspective said chamber of Figure 2, one of the drawers 4 being open. As indicated in FIG. 1, the plate 17 of the drawer 4 is shown in FIG. 4 detached from the support 16 which constitutes the base of the drawer 4 and which fits into two slides (not shown) of the chamber 2.

  Figure 4 shows schematically in perspective, said chamber 2 of Figure 2, one of the drawers 4 being open and the plate 17 being removed. The point of view of FIG. 4 is slightly different from the perspective point of view of the other Figures 1,2, 3 and 30 5.

  28 2849862 FIG. 5 schematically shows in perspective said chamber 2, the top of chamber 2 being removed. There are the respective threaded rods 5 14e, 14f and 14g, of the chamber 2, intended to receive the bolts 14a, 14b and 14c respectively (the threaded rod intended to receive the bolt 14d not being visible). There are also the distinctive elements of the heating glass 18, which are the glass part 18b 10 and the seal 18a.

  The following example serves to illustrate the invention without limiting the scope.

  Example Use of a device according to the invention for the evaluation of the tolerance, recovery and resistance of human keratinocytes subjected to asphyxiation and thermal shock.

  In this example, a stress generating device according to the invention is used, with three drawers such as the device 1 illustrated in FIGS. 1 to 5. The chamber has a total capacity of four liters. The device includes an oxygen sensor, a humidity sensor and a temperature sensor. The% of gas is given by volume.

  Human keratinocytes are seeded at a density of 20,000 cells per well in 200 μl of culture medium defined for keratinocytes as known to those skilled in the art in the central 60 wells of nine 96-well microplates. The peripheral wells of the microplates are filled with sterile water in order to constitute a humidity barrier. Three microplates 5 are placed per drawer in the incubation chamber itself made up of three drawers. Said chamber was previously preheated to 370C using the stress generator device according to the invention. It is also possible to carry out this preheating using a conventional incubator. The chamber is then positioned in a conventional large capacity incubator at 370C under 5% CO 2 and 99% relative humidity.

  Twenty-four hours after inoculation, the 15 keratinocytes are brought into contact with an active principle, or else a cosmetic product dissolved and diluted in culture medium defined for keratinocytes, at different treatment doses. Eight doses of a product repeated six times 20 by microplates are conventionally used, six other wells being reserved for the positive control, that is to say cells without treatment, and the last six wells being reserved for the solvent control. The solvent used is typically ethanol or DMSO (DiMethyl SulfOoxide) which makes it possible to dissolve the non-water-soluble active principles. Each dilution range is repeated on three microplates. It is thus possible according to the invention to test reproducibly three different products in nine microplates in the same incubation chamber. 30 30 '2849862 Twenty-four hours after the treatment of the cells, the stress generating device according to the invention is preheated to the temperature of 37 C. The incubation chamber comprising the microplates of 5 treated keratinocytes is removed from the incubator to be positioned in the stress generator device programmed for a gradual rise in temperature to 430C in fifteen minutes, thus generating thermal stress. In order to subject the keratinocytes for 10 fifteen minutes to a controlled atmosphere under 5% of carbon dioxide, 45% of carbon monoxide and 50% of air (generation of stress by the asphyxiant gas that is carbon monoxide), it is necessary to connect to three separate inlets a bottle of carbon dioxide, a bottle of carbon monoxide and a bottle of air. The air injection rate is set to 250 ml / min, the carbon dioxide rate is set to 25 ml / min and the carbon monoxide rate is set to 225 ml / min using their needle valve respectively. In order to replace the natural atmosphere inside the chamber, the gas outlet is in the open position during the injection period of the gas mixture. The gas leaving the chamber is collected and then stored in a bottle provided for this purpose. The total renewal of the four four liters of chamber volume is carried out in two to three minutes. Once the oxygen level measured using the probe inside the chamber is stable at 10.5%, the gas mixture has completely replaced the natural atmosphere. The gas outlet and inlet are then closed. After 15 minutes of this gaseous stress, a 31 2849862 mixture of 5% carbon dioxide (flow rate set to 25 ml / min) and 95% air (flow rate set to 475 ml / min) is injected into the chamber for two at three minutes in order to completely renew the atmosphere of said chamber, in other words up to measuring a rate of 21% of oxygen in the chamber. The gas mixture containing the asphyxiant gas is collected at the outlet and stored in a bottle provided for this purpose.

  In addition, the cells can be subjected under an atmosphere of 95% air and 5% carbon dioxide only to thermal shock or under a temperature of 370C (normal temperature for keratinocytes) only to asphyxiation so as to 15 distinguish the effects due to stresses taken separately and to cumulative stresses.

  Optionally, once the normal atmosphere has been restored, the keratinocytes can be put into a recovery period known as the post-incubation period, by transferring the chamber comprising the microplates from the stress generating device according to the invention to a conventional incubator with 370C, under 5% carbon dioxide and 99% hygrometry.

  The respiratory activity of the keratinocytes is then measured for example by the calorimetric test with MTT. In the same context, it is possible to evaluate the expression profile of the keratinocyte genes qualitatively and quantitatively by cDNA biochips.

  32 2849862 MTT or biochip evaluation can be performed at O minute, 15 minutes, 30 minutes, 1 hour, 24 hours etc. after the stressful period. This experimental design makes it possible to assess the tolerance, recovery or even the resistance of keratinocytes to asphyxiation and to thermal shock, as a function of their natural physiological state or of the treatment. The different treatments can thus be classified according to their effectiveness.

33. 2849862

Claims (25)

  1. A cell culture device (1) for performing at least one in vitro test, said device (1) comprising at least one chamber (2) in which there is at least one support means (17), and said device (1) comprising at least one means for regulating at least one operating parameter of said chamber (2) during the in vitro test use of said device (1), said parameter being chosen from the group formed by the temperature, pressure, hygrometry and possibly at least one other possible parameter of a possible medium comprising the cell supported by said support means (17), said device (1) further comprising at least one input means in the chamber (2) of at least one fluid and at least one means for leaving the chamber of at least one fluid, said device (1) being characterized in that said chamber (2) is removable.   2. Device (1) according to claim 1 such that the support means (17) according to the invention is generally chosen from the group formed by microplates with wells, test tubes, micro test tubes, boxes Petri dishes, flasks or culture dishes, possibly themselves supported by at least one tray-type support. 34 2849862   3. Device (1) according to one of claims 1   or 2 such that the possible medium comprising the cell or cells and supported by said support means (17) is, under the conditions of use, in liquid or gel form, and the cell or cells are immersed in said medium.   4. Device (1) according to one of claims 1   to 3 such that the possible medium, comprising the cell or cells and supported by said support means (17) 10 also comprises at least one pollutant, liquid and / or solid, supplied before and / or after the use of the device (1 ).   5. Device (1) - according to one of claims 1 15 to 4 comprising at least one isolation means (10, 18a, 3a, 3c, 3d, 4a, 4c, 4d, 5a, 5c, 5d) sealed from said chamber (2) relative to the environment outside said chamber (2).   6. Device (1) according to one of claims 1 20 to 5 such that the fluid which can be admitted into the chamber (2) generally comprises at least one gas chosen from the group formed by carbon dioxide, air , water vapor, oxygen and nitrogen.   7. Device (1) according to one of claims 1   to 6 such that the fluid which can be admitted into the chamber (2) generally comprises at least one polluting gas chosen from the group formed by exhaust gases, cigarette fumes, asphyxiating gases, corrosive gases, 2849862 toxic gases by inhalation, very toxic gases, rare gases and ozone.   8. Device (1) according to one of claims 1 5 to 7 such that the temperature in the chamber (2) is regulated by at least one regulating means (10, 18a, 3a, 3c, 3d, 4a, 4c, 4d, 5a, 5c, 5d) of temperature comprising at least one means of thermal insulation (10, 18a, 3a, 3c, 3d, 4a, 4c, 4d, 5a, 5c, 5d) from the middle chamber (2) exterior 10 and at least one temperature measuring means, associated with at least one adjusting means (10).   9. Device (1) according to one of claims 1   to 8 such that the oxygen content in the chamber (2) is generally regulated by at least one oxygen regulating means comprising at least one means for measuring the oxygen level associated with at least one regulating means.   10. Device (1) according to one of claims 1 20 to 9 comprising at least two chambers (2), only one (2) of which can be used at the same time within said device (1).   11. Device (1) according to one of claims 1 to 10 such that the volume of said chamber (2) is from one milliliter up to ten liters.   12. Device (1) according to one of claims 1 to 11 comprising at least one fluid inlet means, per fluid capable of being used in said device (1).   36 2849862 13. Device (1) according to one of claims 1 to 12 comprising at least one fluid outlet means, by fluid capable of being used in said device (1).   14. Device (1) according to one of claims 1 to 13 such that the various input means possibly present in the device (1) are combined together in a mixing means itself combined with a single input means so that only one mixing inlet means is connected to the chamber (2).   15. Device (1) according to one of claims 1 * 15 to 14 such that a single outlet means is present and directly connected to the chamber (2).   16. Device (1) according to one of claims 1 to 15 comprising at least one means for supplying said chamber (2) with water vapor.   17. Device (1) according to claim 16 comprising at least one water storage means.   18. Device (1) according to one of claims 1 to 17 comprising at least one means for irradiating at least part of the chamber (2) with photons and UV.   19. Method for carrying out at least one in vitro test, in which at least one cell is placed on at least one support means, said support means being placed within at least one chamber (2), said cell being subjected to at least one parameter chosen from the group formed by temperature, pressure, hygrometry and possibly at least one other possible parameter of a possible medium comprising the cell, and to at least one fluid generally comprising at least a gas chosen from the group formed by carbon dioxide, air, water vapor, oxygen and nitrogen, and at least one polluting gas chosen from the group formed by exhaust gases, cigarette smoke, asphyxiating gases, corrosive gases, toxic gases by inhalation, very toxic gases, rare gases and ozone, said method comprising the use of the device according to one of claims 1 to 18 .   20. The method of claim 19 such that, before and / or after carrying out the test, the cell is subjected to at least one parameter chosen from the group formed by temperature, pressure, hygrometry and optionally at least another possible parameter of a possible medium comprising the cell, and at least one fluid generally comprising at least one gas chosen from the group formed by carbon dioxide, air, water vapor, oxygen and nitrogen.   21. Method according to one of claims 19 or 20 such that, before and / or after carrying out the test, the cell is subjected to at least one analysis of at least one parameter. -8 2849 862> 38   22. Method according to one of claims 19 to 21   comprising at least one cell culture.   23. Method according to one of claims 19 to 21   including at least one cell stress study.   24. Method according to one of claims 19 to 21   comprising at least one cell culture and / or at least one cell stress study in the presence of at least one polluting gas, generally chosen from the group formed by exhaust gases, ozone, and cigarette fumes, and / or at least one curative or preventive study of a stress of liquid and / or solid health and / or cosmetological pollutant for pharmaceutical and / or cosmetological and / or environmental purpose.   25. Method according to one of claims 19 to 21   comprising at least one of the following operations, on at least one cell: * Evaluation of the efficacy of anti-cancer products * Study in radiotherapy * Evaluation of photo toxicity and photo protection of cosmetic product * Evaluation of dermocosmetic product against pollution * Assessment of tolerance and / or resistance and / or recovery to a polluting gas and / or to cellular stress 39 2849862 * Assessment of genotoxicity, carcinogenesis or mutagenesis of gas origin, liquid or solid * Evaluation of the cytotoxicity of gas or even gas mixtures and polluting gas (s) * Evaluation of the toxicity and / or the effectiveness of insecticide and / or pesticide * Study of thermal shock * Study of hypoxia, hyperoxia or anoxia * Study of aging * Study of ischemia and / or reperfusion * Fundamental study.