EP2247712A1

EP2247712A1 - Method and device for cell culture in the open continuous mode

Info

Publication number: EP2247712A1
Application number: EP09719279A
Authority: EP
Inventors: Bernard Kudla; Pierre-Yves Chesneau; Yann Beaujouan
Original assignee: Eco Solution
Current assignee: METABOLIUM
Priority date: 2008-02-21
Filing date: 2009-02-20
Publication date: 2010-11-10
Also published as: CA2714186A1; IL207653A0; FR2927906A1; WO2009112739A1; IL207653A; AU2009224507B2; US20110003390A1; FR2927906B1; CN101965394B; US9018007B2; JP2011512148A; JP5620278B2; NZ587479A; CN101965394A; AU2009224507A1

Abstract

The present invention relates to the use of cell cultures in the open continuous mode, to a method for selecting static cell variants or cell variants which proliferate in suspension, to a culture substrate and to a device suitable for implementing this method.

Description

Method and device for cell culture in open continuous mode

The present application relates to the implementation of cell cultures in open continuous mode.

In the conduct of cell cultures, there are traditionally distinguished methods of batch culture and methods of continuous culture. In batch culture techniques, culture vessels containing a sterile growth medium are seeded with a fraction of a culture that has previously grown in a parent culture. In order to be able to reproduce the same characteristics, the daughter culture is generally seeded at a given dilution, and then it is required to perform a growth cycle similar to that of the parent crop. In the case of industrial fermentations, it is useful to reproduce the same growing conditions for each cycle that has been started because this makes it possible to predict in advance when the crops are coming to an end or to reach a favorable growth stage. the recovery of a product of interest synthesized or transformed by the cells.

In the field of research, reproducibility between daughter cultures is necessary to obtain reliable and representative experimental data. To obtain this reproducibility, a synthetic culture medium, identical culture supports and standardized culture conditions are commonly used. The cultures are then placed in closed containers under sterile conditions, in an incubator where constant temperature and pressure prevail.

This reproducibility can be further enhanced by the use of automated incubators allowing, for example, to seed synchronously cultures using a robotic arm. This type of incubator can take the form of a closed chamber within which the temperature and the pressure are finely regulated. Once the seeded containers are sealed and cultured for a specified period.

In a batch culture cycle in a liquid medium, it is known that the cells evolve through successive stages of growth. These different growth stages correspond to distinct physiological states, which are a function of the evolution of the composition of the culture medium over time. In general, as long as the cells are few in the culture medium, they exploit the carbon substrates easier to assimilate and prefer a growth metabolism causing them to actively divide. When said substrates are depleted, the cells begin to hydrolyze more complex substrates using more specific enzymes, and activate more complex metabolic pathways to assimilate them. They enter a subsistence strategy. It is often at this stage that cells synthesize storage products, enzymes and antibiotics, some of which are of industrial interest. At the end of the culture cycle, when the medium is exhausted, many cells die while others resist as biofilms, spores, or any other quiescent form specific to the cellular species concerned.

In batch mode, it is possible, for a given culture medium and cell type, to define from basic physico-chemical parameters such as cell density, pH, oxygen, carbon or nitrogen content. , or any other parameter indicative of the stage of growth of the cells, the stage of growth at which the vast majority of the cells are at a given moment. Indeed, if the culture medium is homogeneous, the development of all the cells is relatively synchronous. It is then possible to associate, for example, a cell density value with a more or less rapid growth phase of the cells, in particular via the use of a typical growth curve. It is also possible to make direct observations under the microscope to determine the growth stage from morphological criteria.

Experiments consisting in reproducing a very large number of times successively the same cycle of culture in batch mode, have been described in the literature. These experiments show that one can reproduce cells over very long periods without altering the characteristics or the properties of the cells (Lenski, R. E. and Travisano, M.

(1994): Dynamics of adaptation and diversification: A 10,000-generation experiment with bacterial populations, Proc. Natl. Acad. Sci. USA 91, 6808-

6814).

In continuous culture techniques, on the contrary, the culture benefits from a regular supply of fresh culture medium or a diluent, that is to say a component of said culture medium, so as to maintain the cell growth for a prolonged period. The dilution of the medium is generally carried out according to a preselected regime, which may be periodic or continuous.

The main methods of continuous cultivation are a mode where the fresh medium is added so as to keep the cell density constant around a mean value (turbidostat mode) and a mode where the fresh medium, or a diluent, is introduced from to maintain a physicochemical parameter (pH, C / N ratio ...) around a defined value (chemostat mode).

Paradoxically, attempts in the literature to maintain cultures continuously over very long periods have failed to maintain cell lines for as long as by using batch culture techniques (Dijkuizen, DE (1993: Chemostat used for studying natural selection and adaptive evolution Meth Meth Enzymol.

224, 613-631). This observation can be explained by the fact that, in continuous mode, the cells reach quite rapidly different physiological stages. and are not all at the same stage of growth. Without doubt, the regular supply of fresh medium disturbs the micro-environment of the cells and does not allow to obtain a perfectly homogeneous culture medium. Thus, it appears systematically, after a number of generations, the appearance of static cell variants called "resistance to dilution". These variants occur in the form of biofilms, filaments or quiescent forms (spores, shells, etc.). They form a sub-population that escapes adaptation constraints and sometimes selection agents (antibiotics or other) used in the culture medium. All described continuous mode culture apparatuses favor the appearance of static variants. These variants occupy the inner surfaces of the apparatus and limit its effectiveness (Chao, L. and Ramsdell, G. (1985): The effects of walls on the coexistence of bacteria in the liquid phase of chemostat cultures. Microbiol 131, 1229-1236). Continuous cultures with constant cell concentration (turbidostat mode) are particularly sensitive to invasion by dilution-resistant variants. As a result, they can only be run over relatively short periods, generally for less than 200 generations. On the other hand, the continuous cultures carried out under the conditions where the cell growth rate is high favor spontaneous mutations in greater numbers.

These conditions are particularly favorable for the development of adherent variants developing in biofilms, insensitive to dilution.

Because of these various difficulties, the continuous culture methods and devices disclosed in the literature are little used for industrial and even scientific purposes.

Their potential was however recognized very early (Monod, J. (1950): The technique of the continuous culture Theory and applications, Ann. 79, 390-410; Novick, A. and Szilard, L. (1950): Description of the Chemostat, Science 112, 715-716).

However, continuous culture techniques are now experiencing an unexpected revival of interest precisely because they make it possible to promote the emergence and selection of cell variants with differentiated growth. It may thus be desired to obtain proliferating cell variants in suspension or, conversely, static cell variants. These are particularly useful for studying, for example, the biology of biofilms. The principle of this selection is to take advantage of the spontaneous evolutions favored by the cultures in continuous mode, to select, in the long term, cell variants which evolve preferentially in suspension, or on the contrary mainly in static forms.

Whether or not they result from genetic mutations, suspension-proliferating variants generally tend to divide more actively than other cells present in the medium, or to make better use of the resources of the medium. If the culture is maintained over a long period, the frequency of these variants within the population increases over time, making it easier to isolate them. Suspended growth variants selected according to this principle generally have a competitive advantage over other cells from the same line. They are therefore useful for improving, for example, the yield of existing industrial fermentation processes, such as those practiced in batch culture mode. The international application WO 00/34433 describes a device for the implementation of continuous cultures allowing the selection of proliferating variants in suspension. This device is provided with two culture vessels interconnected by a pipe, which allows to transfer at will a culture contained in a first container to a second and vice versa. This pipe is equipped with a valve which is operated at will to make pass the culture from one container to another. Each of the two culture vessels is fed by an independent fluidic network allowing a constant renewal of medium and gas inside each of the containers. On this rather complex fluidic network, a second fluidic network connects to clean and sterilize each of the two culture vessels, independently, using sterilization fluids. Said device is thus designed to be able to transfer a culture in liquid medium from a first container to a second one. When one container contains the culture, the other can be cleaned and sterilized and vice versa. During sterilization and cleaning, the static variants attached to the walls of the container are removed, while the proliferating cells in suspension are maintained in continuous culture in the other container. The device is designed to repeat the operation as many times as necessary. It is thus theoretically possible to maintain a continuous culture for an unlimited duration while avoiding the accumulation of static variants.

However, in practical terms, this device has several major drawbacks, including the following:

The cleaning and sterilization of the containers within the device require a complex fluidic network. This fluidic network must be perfectly sealed to maintain an adequate level of sterility. However, it is difficult to reproduce such a fluidic network on an industrial scale, because when the volume of the culture vessels is increased, the volume of sterilization and washing liquids necessary for the operation of the system becomes too great. These fluids must be stoked while waiting to be decontaminated, which leads to high maintenance costs. In addition, the operation of the system requires the mobilization of two tanks for an effective selective culture. These elements limit the number of crops that can be implemented on the same technical platform.

Access to cells in culture is difficult because of the fact that the cultures are kept in a closed container. This access is all the more reduced, it must be ensured the sterility of the fluidic network. It is therefore difficult to monitor crops, especially real-time monitoring.

The introduction into the culture of non-soluble substances, immiscible or difficult to miscible with the medium used, is also complicated by the need to borrow the fluidic network and to respect the confinement of cultures.

The pressure between the different compartments of the device is difficult to regulate, making it virtually impossible to maintain a constant and homogeneous pressure within the system. Under these conditions, the reproducibility of the crops is affected. Moreover, overpressures in the fluidic network can occur and thus disrupt or interrupt the operation of the device;

The passage of the culture from one culture container to the other does not make it possible to perform cultures in a static mode because it necessarily involves a resumption of the cells in suspension and the elimination of the static variants.

International application WO 2005/083052 describes another device for the selection of proliferating variants in suspension in continuous culture. This device takes the form of a flexible tube, inside which the culture is produced between two clamping points located upstream and downstream of the tube. The renewal of the culture medium is carried out by adding fresh medium and the elimination in the same volume of the spent medium by moving the tube between the clamping points. The opening and closing of the clamping points creates a peristaltic effect. The new tube segments introduced between the clamping points bring the medium fresh and participate in the dilution, while the static mutants, which are fixed on the walls, are eliminated as the tube is displaced at the level of the used segments. of it.

This device has the same limitations as the previous one with regard to the volume and confinement of the culture. In addition, it seems difficult to ensure proper gas exchange by diffusion through the single wall of the tube.

The method and apparatus of the present invention are intended to overcome the limitations of the above-described culture devices.

One of the objectives of this invention is to allow continuous culturing of cell cultures in order, in particular, to practice the selection of variants having a determined growth stage. According to the invention, these cultures are carried out in culture supports and culture vessels in open mode, placed in a closed enclosure.

By culture vessel, or culture vessel is meant according to the invention a container of the culture medium, generally intended to be brought into direct contact with the culture medium. By culture support is meant according to the invention an assembly comprising a culture vessel and a support of the culture vessel. The culture support described hereinafter according to the invention is itself a particular culture support.

The particular structure of the continuous cell culture method described hereinafter, whatever the culture vessel (s) used and placed inside the closed chamber used in this process, allows easy access to the cultures produced in continuous mode. .

The particular structure of the culture media according to the invention described below, placed inside the enclosure of the device according to the invention, allows easy access to the cultures produced in continuous mode. These culture supports are designed so that the culture vessels they comprise can be replaced at will, for example according to a preferred aspect of the invention when the cultures accumulate too many static variants. But other culture media and / or culture vessels may also be considered in the context of the present invention, and used in the method according to the invention. Manipulations inside the closed chamber can be performed using robotic manipulator arms, which also considerably reduces the risk of contamination.

According to the invention, the number of culture supports and / or culture vessels present in the device can be adapted by simple dimensioning of the closed enclosure where they are placed. It is therefore possible to implement several crops at once, in series or in parallel.

The method and the device, as well as the method for implementing this device, described in the present application, have numerous advantages, among which we can mention:

The possibility of cultivating solids or oligo-cellular organisms in suspension (aerobic or anaerobic according to the gas mixture present inside the closed enclosure), at homogeneous temperature and pressure, in a liquid phase, under sterile conditions. In particular, that of cultivating photosynthetic microorganisms which require constant illumination and a culture mode in suspension, without agitation;

The possibility of performing continuous cultures on a large number of constant volume generation, at a cell growth stage defined according to a predefined physico-chemical parameter measured directly in the culture (cell density, pH, concentration of a product ...) over a period that can be very long.

The possibility of measuring the different parameters required for online monitoring or monitoring of the crop (pH, pO2, substrates, products, etc.).

The possibility of applying to the culture physico-chemical parameters that create a selection pressure and that can be modulated (inputs of molecules, T ⁰ C, pH, ...)

The possibility of separating as frequently as necessary, the biomass in suspensive growth (subject to dilution) of immobilized biomass on any wall of the culture system (insensitive to dilution), by pipetting and transfer of the culture into a new culture vessel sterile and / or replacement of the culture medium. The possibility of carrying out a selection of the static variants (instead of the proliferating variants in suspension) by evacuation of the liquid phase present at the level of the culture vessels and / or supports and replacement of this liquid phase with a fresh medium by proceeding, by means of for example, resuspending static variants attached to the support.

The possibility of using culture media and / or culture vessels and / or disposable pipetting equipment, disposable or recyclable.

The present invention relates to a generally open continuous mode cell culture method, in particular allowing the selection of proliferating cell variants in suspension or in a static manner.

By culture in continuous mode, or continuous culture, denotes according to the invention a culture carried out in a liquid medium and a fraction of said culture medium is renewed in order to maintain the cells growing in a prolonged manner. Preferably, the cells are maintained for a large number of generations which is not defined in advance, preferably greater than 100 generations, more preferably 200, even more preferably 1000 generations.

The renewal of the culture medium, or a component thereof (diluent), can be carried out permanently, regularly or periodically. The culture medium can be renewed for one or more of the ingredients in its composition, or for the entire mixture of these ingredients. The culture medium is generally renewed so that at least a majority of cells, preferably at least 50% of the cells in culture, more preferably at least 80% of them are kept in suspension. According to the invention, the culture medium is more generally a liquid culture medium. Subsequently, the terms "culture medium" and "culture" are used interchangeably. A "cell" is defined here as a small biological entity comprising a cytoplasm delimited by a membrane and having the capacity to reproduce autonomously. The cell may be eukaryotic or prokaryotic, animal or plant. Microorganisms are considered cells.

Bacteria, yeast and unicellular algae are preferred cells for carrying out the present invention.

By liquid culture medium is meant a liquid mixture comprising nutritive substances, as well as optionally other components such as selection agents (antibiotics), in which cells can multiply.

A cell variant is defined as a daughter cell that does not have the same physiological characteristics as its parent cell grown under the same conditions. Physiological changes of which the variant is the subject may occur due to genetic modification (point mutation, loss or acquisition of genetic material), but may also result from stress or other factors that may have a lasting impact on the behavior of the cells in culture. It is not required according to the invention that these physiological modifications are predefined. On the contrary, the object of the invention is to promote the emergence of cellular variants from spontaneous modifications, then to select among these variants those having acquired properties conferring on them a competitive advantage over the other cells in culture. In general, these new properties allow them to multiply more quickly or to better exploit the culture medium.

The cellular variants according to the invention can be selected according to two main modes: suspension variants: variants which are more competitive in a planktonic development in liquid culture are selected. In this case, it is the static variants that one seeks to eliminate. Static variants: Variants that bind, aggregate, encyst, or take any other form of dilution resistance, for example in the form of biofilms, are selected. In this case, it is the variants that develop in suspension that one seeks to eliminate. The continuous cell culture method allowing the selection of static cell variants or proliferating in suspension according to the invention is characterized in that it comprises one or more of the following steps, more generally the following steps, by which: a) is seeded with the aid of one or more living cells, a liquid culture medium contained in a first culture vessel kept open in a closed chamber, b) feeding said cells, in said culture medium, to a determined growth stage, corresponding to a given cell density or to a physico-chemical parameter measured in the culture medium, c) the cell density in the culture medium, or the value of said physico-chemical parameter, which is reached in step b) is kept substantially constant by adding fresh culture medium or at least one diluent to said culture vessel, d) pipetting a portion of ie the culture medium obtained in c) containing the cells in suspension so as to maintain the volume of the culture; e) transferring a fraction of the culture medium obtained in d) into a second culture vessel, which generally replaces the first culture vessel; f) removing or eliminating said first culture vessel with the remaining culture fraction contained therein; g) after several generations of culture in the second vessel, the suspension-proliferating cells and / or the static cell variants are selected.

In step c) according to the invention, whether for the process according to the invention above or for one of the processes according to the invention described above. after, it is not excluded that the addition of fresh culture medium or at least one diluent in said culture vessel is also a simultaneous supply of fresh culture medium and at least one diluent. In general, by at least one diluent, a (single) diluent is preferred, but it is possible to use several diluents without departing from the scope of the invention.

According to the invention, all the culture vessels present in the closed chamber are generally identical, but it is possible that the culture vessels are different from each other, or are of several different types within the same enclosure. According to this method, it is possible to select, separately or simultaneously, suspension-proliferating cell variants as well as static variants.

When it is more particularly desired to select suspension-proliferating variants, it is more particularly possible to proceed in the following manner. A method according to the invention of continuous cell culture for the selection of cell variants proliferating in suspension, can be characterized in that it comprises the following steps: a) one or more living cells are seeded with liquid culture medium contained in a first culture vessel kept open in a closed chamber, b) said cells are brought into said culture medium at a determined growth stage, corresponding to a given cell density or to a physiological parameter. measured in the culture medium, c) the cell density of the culture, or the value of said physico-chemical parameter, reached in step b), is kept substantially constant by a supply of fresh culture medium or at least a diluent in said culture vessel; d) pipetting a portion of the culture medium containing the cells in suspension to maintain the volume of e culture; e) transferring a fraction of the culture obtained in d) in which the cells are suspended, in a second culture vessel replacing the first; f) removing said first culture vessel with the remaining culture fraction contained therein; g) after several generations of culture in the second container, the cells proliferating in suspension are selected.

In this aspect, the fraction of the culture removed with the container in step f) is generally comprised of static variants. Step f) is a removal step, or an elimination step, or a withdrawal or removal step.

When it is more particularly desired to select static variants, one can proceed as follows: a) one inoculation with the aid of one or more living cells of a liquid culture medium contained in a first culture vessel in which is placed at least one solid surface, preferably a plate, said container being kept open in a closed chamber, b) the cells are brought into the seeded culture medium at a determined growth stage, corresponding to a given cell density, or to a physico-chemical parameter measured in the culture, c) the cell density of the culture, or the value of said physico-chemical parameter, reached in step b), is kept substantially constant by a supply of fresh culture medium or of at least one diluent in said culture vessel, d) pipetting part of the culture medium obtained in c) containing the cells in suspension so that maintain the volume of the culture; e) transferring said solid surface on which a fraction of the culture obtained in d) is deposited, in a second culture vessel replacing the first; f) removing said first culture vessel with the remaining culture fraction contained therein; g) the static cell variants attached to said solid surface are selected after several generations of culture.

According to this aspect of the invention, the fraction of the culture removed with the container in step f) most often corresponds to a liquid fraction in which most of the proliferating cells are present in suspension.

Step f) is a removal step, or an elimination step, or a withdrawal or removal step.

This embodiment of the invention more particularly involves solid surfaces. These solid surfaces provide the opportunity for static cell variants to bind during the selection process. Solid surfaces can take different forms such as plates, beads or particles. They can be made of different materials (plastics, metals, glasses, minerals, composites), inorganic or organic. Plastic materials such as polystyrene, polycarbonate, polyethylene, polypropylene, polyurethane and their derivatives are preferred. Surfaces can be treated physically or chemically. They can be suspended in the culture medium, hooked or placed at the bottom of the culture vessel. Preferably, the solid surfaces are designed to be removable through the opening of the culture vessel, and placed in a new culture vessel including, for example, fresh medium.

According to a preferred embodiment of the method, said solid surface is made of a material treated to prevent the adhesion of the cells. Indeed, the method may include a step in which different surfaces are tested to determine which of these surfaces provides better or lesser adhesion of the static variants. In this, the method may be particularly useful for the selection of cell adhesion limiting materials for the purpose of, for example, developing surgical equipment such as catheters limiting contaminations.

Conversely, this method can be useful for selecting materials promoting the installation of cells (biogenic surfaces) sought in the development of prostheses or medical implants.

The method according to the invention advantageously allows the selection of cell variants at a predefined growth stage. Indeed, according to step b) of said method, the cells are preferably brought into the culture medium at a growth stage, which is determined or linked to the cell density or to a measurable physicochemical parameter in the culture medium. such as pH, amount of dissolved oxygen, available carbon or nitrogen, etc. To achieve this "determined" growth stage, one can rely on typical growth curves, established at in advance, experimentally or from literature data. These curves are generally established on the basis of cultures carried out in batch mode. They make it possible to relate the cell density or a physico-chemical parameter of the culture medium to the physiological state in which most of the cells in culture are found at a given moment.

It is known to those skilled in the art that the cells during a culture cycle pass through different physiological states, in particular linked to the depletion of the culture medium in certain substrates, in particular when the medium is not renewed. of culture. These physiological states generally reflect an adaptation of the cells vis-à-vis their environment.

According to a preferred aspect of the invention, a particular value of a measurable physico-chemical parameter is set, for example, a cell density value known to be a parameter determining the secretion of an enzyme of interest. The method provides that after inoculation of the culture medium, the cells grow to reach the set value. When this critical value is reached, the continuous culture is started so as, for example, to keep the cell density constant. It is thus possible to keep the cells as long as possible in the desired physiological state, which makes it possible, for example, to prolong the period during which the cell will secrete the product of interest. According to the invention, the cell cultures are generally carried out in an open mode, which facilitates the interventions and the samplings required to maintain the physicochemical parameters chosen at a constant value. This means that the culture vessels chosen to implement the method usually have a sufficiently wide and practical opening, preferably oriented upwards or vertically, to be able to advantageously introduce the material necessary to carry out samples of culture medium. by pipetting or direct measurements using probes. By vertex is generally meant the highest point of the culture vessel, relative to a horizontal base which may be the ground.

Thus, according to a preferred embodiment of the invention, the method is characterized in that said culture vessels are kept open at their top, and that a gaseous flow, such as sterile air, is applied in such a way that continues, on the outskirts of their opening.

In this, the present process is distinguished from the continuous or semi-continuous culture processes described in the prior art, which are carried out in closed containers, which do not allow to establish a real-time monitoring of crops. The method according to the invention therefore makes it possible to carry out a selection of cell variants maintained, or even synchronized, at a predefined growth stage. This is particularly useful for selecting, for example, variants of cells that synthesize products of interest transiently, such as enzymes or antibiotics. The selection of cell variants can thus be implemented by reproducing the conditions in which the cells synthesize the product of interest. The process according to the invention is therefore particularly suitable for the improvement of industrial strains used in fermentation processes, in particular those used in semi-continuous mode (that is to say those during which the culture medium is renewed on a continuous basis. fixed duration).

A particular aspect of the invention consists, independently of the selection of suspended variants or of static variants, in the implementation of process of the method described above to synthesize a product of interest for an unlimited period, maintaining the cells at an optimal growth stage for the synthesis of said product.

The method according to the invention is generally implemented in a closed chamber, the size of which may vary according to the needs of the users, the number and the volume of the crops.

More particularly, according to one embodiment of the invention, the various steps a) to f) are performed in a closed enclosure.

The pressure and the temperature can be kept constant in the enclosure at the desired values. Since the culture vessels are open, the cultures are generally at the same pressure as that applied in the enclosure. The risks of local overpressure encountered in the confined systems of the prior art are thus generally eliminated.

Said enclosure also makes it possible to control the gaseous environment of the cultures, which is particularly useful in the case of culture carried out under anaerobic conditions.

The culture method most often provides that sterile gas, such as sterile air, is injected under pressure into the cell culture medium by means of a bubbling device, for example in the form of aeration rods. introduced into the culture vessel generally through the opening of said container. This gas injection aeration the culture medium, the homogenization of said medium by air lift (agitation bubbling) and helps maintain a certain gas pressure inside the chamber.

According to the method, the enclosure is preferably traversed by a sterile gaseous flow. This stream may consist of a gas, such as nitrogen or a mixture of gases such as air, depending on the chosen culture conditions. Preferably, the sterile gaseous flow is applied at the periphery of the opening of the open culture vessels to remove contaminants from this zone and thus reduce the risk of contamination. This flow also makes it possible to balance the pressure inside the enclosure.

The circulation conditions of the sterile gaseous flow are most often the same for all the containers of the closed chamber. For greater efficiency of the system, the gas flow, which is preferably a laminar flow, is generally applied either from top to bottom of the culture vessels or from the bottom to the top of the culture vessels, generally outside said containers. of culture. The device according to the invention described below is a device in which is applied (or directs) the sterile gaseous flow from top to bottom of the culture vessels.

More preferably, the gas flow is activated at the periphery of the culture vessels, in particular around the opening of said containers, creating a depression at the bottom of the culture vessels. In order to obtain this depression locally, the culture vessel may be placed in an upwardly open confinement tank provided with suction and evacuation means for the gas flow at its base. A depression can then be obtained locally in the volume located between the culture vessel and the internal walls of said containment tank. Preferably, the sterile air travels the volume between the inner walls of the containment vessel and the culture vessel from top to bottom, and is evacuated from the enclosure at the base of the culture vessels. In this way the contaminants are trapped in said volume and driven to the lower part of the containment tank. Thus, they do not penetrate inside the culture vessel, which is slightly overpressurized, especially because of the supply of gas produced by bubbling in the culture medium.

Another embodiment would include the activation of the sterile gaseous flow from bottom to top of the culture vessels.

According to a preferred embodiment of the invention, several cultures are carried out simultaneously in several culture media placed in the same enclosure. By many we mean at least two.

The sterile gas stream is particularly useful for avoiding cross-contamination when different crops are grown simultaneously in the same enclosure. According to a preferred aspect of the method described above, steps a) to f) above are repeated one or more times before proceeding to step g). According to one embodiment of the invention, the maintenance of a substantially constant cell density in the culture in step c) is carried out by dilution of the culture with fresh medium while maintaining a constant volume of culture medium. in the culture vessel. In the case of selection of suspended variants, the transfer of the culture medium containing the cells in suspension into the second culture vessel can be performed by pipetting with a sterile pipette. The pipetting operation is preferably carried out using a robotic arm located inside the closed chamber. The first culture container used, is generally removed from the culture medium to which it can belong. In all cases, this first culture vessel is evacuated outside the closed chamber with the aid of an airlock. This airlock keeps pressure and sterility stable inside the enclosure. The evacuated container is usually removed. The culture vessel in operation can be placed in a thermostated cell, that is to say in an open chamber whose walls are maintained at the desired temperature, allowing the regulation of the temperature of the cell culture. In a preferred aspect, the containment tank is itself thermostated and serves as a cell. One of the walls of the tank may, in fact, comprise a heating means for regulating the temperature of the objects placed in the interior volume of said tank. In the case of the presence of a thermostated cell around the culture vessel, the space between the culture vessel and the inner walls of the containment tank must of course be understood as the space between the thermostated case and the tank confinement.

The culture supports and / or the culture vessels according to the invention are preferably disposable and compatible with robotic manipulation inside the enclosure. Indeed, the invention preferably provides that at least some, that is to say several, process steps are performed using one or more automated arms allowing movement within the pregnant. Preferably the enclosure remains closed during the various steps of the process. According to the invention, the cells are generally cultured continuously over a number of generations greater than 10 ² , preferably greater than 10 ⁴ , more preferably greater than 10 ⁶ , and still more preferably greater than ¹⁰ generations, without opening (direct ) of the enclosure on the outside environment.

Preferably, the culture method according to the invention in open continuous mode is implemented using a culture device particularly suitable for this purpose.

The invention also relates to a culture support for performing an open continuous cell culture, characterized in that it comprises: at least one culture vessel open at its top suitable for containing a liquid culture medium; at least one upwardly open containment tank in which said culture vessel is housed; a space between said culture vessel and said containment tank, adapted to circulate a gas flow at the periphery of the container opening, from top to bottom, between the culture vessel and the inner walls of the containment tank; and a means for extracting said gaseous flow, located in the lower part of the containment tank.

Preferably, the means for extracting the gas flow consists of one or more orifices allowing the gas flow to pass, for example air.

In one embodiment, the culture support is in the form of a removable block in which several of said containment bins are grouped and in which are housed at least one of said culture vessels.

Preferably, said lower portion of the containment tank is flush with a base provided with complementary extraction means of said gas flow flowing between the culture vessels and the walls of the containment tanks, such as a pipe connected to a pump empty, or one or more gas distribution means. The culture support according to the invention may comprise at least one means for regulating the temperature of the internal volume of said containment tank, said means for regulating the temperature of the internal volume of said containment tank preferably consisting of a heating resistor included in at least one of the walls of said containment tank. The culture support according to the invention may comprise at least at least one means for injecting air into the medium (s) of culture, contained in the said container (s). ), such as one or more aeration rods.

The culture support according to the invention may comprise at least one means for optical measurement of the density of the cells present in the culture medium contained in the culture vessel.

The culture support according to the invention may comprise at least one means for producing light for the cultivation of microorganisms in autotrophic mode, located in the space between the culture vessel and the internal walls of said containment tank, or included in one of the walls of said containment tank.

The invention finally relates to a cell culture device for continuous growth of cells in open mode, characterized in that it comprises: - an enclosure; means for generating a sterile gaseous flow flowing through said enclosure;

- One or more culture media according to the invention as described above, positioned in said chamber, for performing a cell culture in open mode.

The cell culture device may comprise at least one means of renewal of the culture medium placed inside said chamber.

The cell culture device may comprise at least one culture vessel adapted to replace that contained in the culture support.

The cell culture device can be characterized in that the means for generating the sterile gaseous flow is placed in the part upper chamber so that said sterile gas stream is directed from the top to the bottom of the culture support.

The cell culture device may further comprise at least one means for extracting the sterile gaseous flow at the base of said culture vessel, said means for extracting the sterile gaseous flow preferably creating a vacuum in the space between the culture vessel and the containment vessel of the culture support. Said extraction means may be associated with at least one air suction means adapted to suck the air surrounding the periphery of the opening of the culture vessel. The cell culture device may be characterized in that the culture medium renewal means comprises means for transferring part of the contents of the culture vessel to an evacuation zone.

The cell culture device may be characterized in that the culture medium renewal means comprises means for transferring fresh medium from a reserve located inside the chamber to the culture vessel.

The decanting means preferably comprises a pipette and a suction means of fresh or used medium in said pipette. The cell culture device may further comprise at least one outlet means to the outside of the enclosure of the transfer means and / or at least one culture vessel, said outlet means preferably comprising an airlock .

The cell culture device may be characterized in that it comprises at least one means for injecting air into the culture.

The cell culture device may be characterized in that it comprises at least one automated arm adapted to make displacements within the enclosure.

The cell culture device may be characterized in that the enclosure is closed. The cell culture device may comprise a plurality of culture supports positioned in said chamber, to perform in parallel several open-mode cell cultures.

The cell culture device may be characterized in that it allows the implementation of the method according to the invention.

The invention will be better understood on reading the following figures, among which:

Figure 1 schematically shows a first culture support according to the invention, in perspective;

Figure 2 shows schematically a second culture medium according to the invention, in perspective;

Figure 3 shows schematically a removable block having cells for culture media as shown in Figure 2, in section III - III with respect to Figure 4;

Figure 4 shows a removable block having cells of Figure 3, in plan view;

Figure 5 shows schematically and partially a cell culture device according to the invention, in perspective; Figures 6 to 10 each schematically represent an operating plan of a continuous cell culture method according to the invention, in a closed chamber (not shown) using any culture medium, each corresponding Figure specific steps of the Figure 7 showing initial steps of a cell culture;

- Figure 8 showing steps of changing the culture vessel; and

- Figure 9 showing steps of harvesting culture medium; and

- Figure 10 showing micro sampling steps for analysis. Figure 1 shows schematically a first culture support 1 according to the invention, open upwards, suitable for containing a liquid culture medium. The support 1 comprises a culture vessel 6 housed in an upwardly open confinement tank 2 in which the said culture vessel is housed. The culture vessel is placed in a thermostated case 3, which is optional according to the invention.

The culture medium (shown in fill - dashed lines - in FIG. 1) is capable of being fed with gas, for example air, most often under pressure, by a bubbling device constituted in this case by a ventilation rod. 4 in the form for example of a bubbling cane. This rod 4, held by means of an arm 5, is immersed vertically in the culture vessel 6, where the culture medium is located, by the opening of the culture vessel 6. The arm 5 can carry out a vertical movement to position the lower end of the rod 4 at the desired location in the culture vessel 6.

The hollow arrows pointing downwards in FIG. 1 symbolize the sterile gaseous flow, which is for example sterile air, which flows from top to bottom and which passes through the space, or volume, 7 situated between the inner wall of the containment tank 2 and thermostatic case 3. The sterile gaseous stream is discharged through several discharge openings 8. The discharge openings 8 represent a means of extracting the gas stream, and are located in the lower part, of Preferably the base of the container containment 2. The culture vessel 6 may consist of any type of upwardly open container, such as vial, bottle or Erlen. It is preferable that the culture vessel 6 can be easily positioned and withdrawn from the containment vessel 2. As shown in FIG. 1, it is advantageous to choose a culture vessel 6 which does not exceed the height of the walls of the vessel. containment tank 2.

The sterile gaseous flow whose flow can be organized from the top to the bottom of the culture support 1, is designed to create a barrier aseptically around the opening of the culture vessel 6, delimited by the internal walls of the containment tank 2.

This gas flow can be activated by creating a depression in the lower part of the containment tank 2, for example by connecting the discharge ports 8 to a suction means.

The lower part of the containment tank 2 is preferably designed to fit on a base provided with complementary extraction means of said sterile gas stream, such as a pipe connected to a vacuum pump. Where appropriate, said base may be provided with one or more gas stream collection or distribution means, useful for implementing the continuous mode culture method.

Figure 2 schematically shows a second culture support according to the invention, 1 ', in perspective. The culture support 1 'comprises a culture vessel 6' which is present in a containment tank 2 ', represented here in transparency. The culture vessel 6 'and the containment vessel 2' are both open upwards. The culture vessel 6 'is able to easily position and withdraw from the containment tank 2', because it does not exceed the height of the walls of the containment tank 2 '. The culture vessel 6 'comprises a culture medium 11.

A space T is defined between the culture vessel 6 'and the internal walls of the containment tank 2', said volume 7'apte to be traversed by a gaseous flow from bottom to top. The discharge means of said sterile gas stream are not shown here, but they consist of several orifices located in the base of the containment tank 2 '.

Two means for optical measurement of the density 9 of the cells present in the culture medium are placed on either side of the transparent culture vessel 6 ', and in the space 7'. Inside the culture vessel 6 ', a vertical inner wall 10 is present. It allows a better circulation of the injected gas in "air lift" in the culture medium 11 through the aeration rod 4. Figures 3 and 4 show a preferred culture support 1 "according to the invention in the form of a removable block 12, comprising six cells, or compartments, 24, 25, 26, 27, 28 and 29, in which are inserted Figure 4 shows the block 12 in top view, and Figure 3 shows the block 12 in cross section III - IM (see Figure 4).

The block 12 comprises four culture vessels 6 ', themselves having walls 10, and each housed in a cell 25, 26, 28 and 29 of the block 12. Each culture vessel 6' can be placed manually in a cell. Block 12 is preferably made of a part. Block 12 is in fact as if several culture supports 1 'as shown in Figure 2, and described above, were contiguous to the outer wall of their containment bins 2'. The cells are provided with walls at their periphery forming containment tanks 16. Gas distribution means, collector and filter contained in a ventilation module 13 are extended by individual aeration rods 4 to reach each of the culture vessels 6 '. The sterile gaseous flow through the culture support 1 "from top to bottom is symbolized by hollow arrows, while the path of the gas supplying the culture media is represented by solid arrows and gas bubbles. sterile, preferably permanent, ensures the confinement of each culture vessel 6 'when the block 12 is placed in a closed enclosure.This culture block 12 can be thermostatically controlled by the presence of heating resistors incorporated in its mass. is evacuated at the base of each of the cells having a culture vessel 6 'by means of evacuation 14 in the form of pipes intended to be connected to an air suction means such as a vacuum pump.

In its low central part, the block 12 preferably comprises a collector and distributing ramp of the aeration gas or gases. These gases are conveyed inside each culture vessel 6 'by means of a detachable bent tubing 4, terminated in a rod 4 at the end which is immersed in the culture vessel 6 ', and which can be manually positioned on the distributor rail at connectors, after the installation of the culture vessels 6'. These bent pipes bring the gases into each culture vessel 6 'in the lower position to cause an "air lift" for ensuring the supply of oxygen, carbon dioxide or other gas (s) in the culture medium and to ensure a homogeneous mixture of the culture medium.

Bright side panels, or illuminating plates, 15 are arranged in two cells 25 and 26, laterally, to allow the culture of photosynthetic microorganisms. The plates 15 are preferably removable and are light production panels for the culture of photosynthetic microorganisms, and are located in the space between the culture vessel 6 'and the inner walls of the containment tank 16, or included in one of the walls of the containment tank 16. Preferably, the light is produced using LEDs, the wavelength of which is chosen according to the photosynthetic pigments of the microorganisms concerned.

The light emitted may correspond to white light or to lights of different wavelengths depending on the type of LED used. In addition, a stroboscopic lighting regime is achievable depending on the mode of operation of the LEDs. These lighting possibilities allow the cultivation of photosynthetic microorganisms such as prokaryotic or eukaryotic microalgae, especially in autotrophic mode, especially by combining lighting and the use of CO2 in the aeration and mixing gases introduced into the cultures.

By autotrophic mode is meant a culture in which the cells produce organic matter by carrying out the reduction of inorganic material, for example in the form of carbon dioxide, and by removal of inorganic salts in the medium. The energy required for this synthesis comes from light, as in the case of photosynthesis for example. The culture support according to the invention thus makes it possible, if necessary, to illuminate the cells continuously at each of the steps of the process according to the invention.

According to a variant not shown, the light generating means may also take the form of a panel forming all or part of one of the walls of the containment tank.

According to one particular aspect of the invention, the means for producing light consist of lamps or UV diodes whose function is either to decontaminate the interior volume of the confinement tank by irradiation before or after use, or to generate mutations on the cells in culture during the selection process.

Optical measurement means 9, which are measuring forks of the turbidity of the culture medium, are inserted, in pairs, in the lower part of each of the cells 24, 25, 26, 27, 28 and 29 to follow the evolution. the cell density of the culture media present in these cells.

The culture supports in the form of blocks 12 described above have the advantage of facilitating the implementation of the continuous culture method according to the invention. Indeed, several culture vessels 6 'can be prepared and placed in a sterile chamber before the start of operations. The connection of the distributor ramp to the system for supplying gases inside the enclosure is done by a quick coupling when a block is positioned on a specific base of the culture device according to the invention, which can besides hosting several of these blocks. Similarly, it is expected that the necessary electrical connections (heating, detectors, lighting) are made when placing a block on a base by simple connection. Such a block facilitates pre-operative handling and, subsequently, reduces the automated movements that take place inside the closed enclosure during the implementation of the culture method. The device can then operate autonomously over a longer period.

According to a preferred embodiment of the invention, the culture device is an automaton that can comprise a number of removable blocks 12 to six tanks which can vary from one to 100. Each of the blocks 12 is manually conditioned, sterilized and introduced into the controller on a base provided for this purpose. When the blocks 12 are installed, the selective culture process can start by filling a culture vessel, its inoculation, and then producing a continuous culture at a constant volume according to the method already described. It is thus possible to produce in parallel a large number of continuous cultures, each block 12 being the place of an independent experiment.

When a culture requires its transfer, for example because the accumulation of static variants becomes too great, a culture volume of a first culture vessel of the block 12 is pipetted and transferred to a second culture vessel of the same block. When all the culture vessels of a block 12 have been used, the culture can be transferred to a culture vessel of another available block 12, while the fully used block can be replaced by a new conditioned block provided with six new sterile culture vessels.

The use of the device provided with culture supports in the form of blocks also makes it possible to simplify the robotic functions of pipetting, dilution and transfer. A device according to the invention can carry out the selective cultivation of a single microbial species or of a given microbial consortium in ten, twenty or at least forty blocks, ie 60, 120 or at least 240 tanks simultaneously in order to reach a total volume. cultivation up to 1200, 2400 or at least 4800 mL. In this way, the probability of obtaining a given mutant increases accordingly. This increase is particularly interesting for cells with a complex genome or for those of slow-growing species.

Figure 5 shows a continuous culture device 100 according to the invention. An enclosure 17, which is closed when the device 100 is in operation, is suggested but not shown completely.

The device 100 comprises several culture supports 1, as represented in FIG. 1, the enclosure 17 and a stream generation means. gaseous sterile 23 passing through said enclosure 17. The culture media 1 operate in parallel in the enclosure 17. The enclosure 17 is traversed by a robotic arm 19 mounted on a rail, capable of moving in the three dimensions of space . This robotic arm 19 is equipped with a transfer pipette 20 for the renewal of the culture medium, a dilution pipette 21 and a handling clamp 22.

The size of the chamber 17 is not limited, which allows several cultures to be carried out simultaneously in several culture media, identical or different, placed in the same chamber 17. The sterile gas flow through the chamber 17 is represented by hollow arrows from the means 23 located in height of the enclosure 17 so that said sterile gaseous flow is directed from the top to the bottom of the culture support 1. The sterile gaseous flow which passes through the enclosure 17 is evacuated outside the enclosure 17 through orifices 8 situated at the base of the culture supports 1. Preferably the means for extracting the sterile gaseous flow is designed so as to create a negative pressure in the space situated between the culture vessel 1 and the containment tank 2 of the same culture support 18. It is generally associated with a gas flow suction means adapted to suck up the gaseous flow surrounding the periphery of the opening of the container. culture, such as a vacuum pump (not shown) located, for example, under the floor of the enclosure 17.

Figures 6 to 10 each schematically represent an operating plan of a continuous cell culture method according to the invention, in a closed chamber (not shown) using any culture support, each Figure corresponding to a functional diagram explaining specific stages of the operation,

Figure 7 showing initial steps of a cell culture; - Figure 8 showing steps of changing the culture vessel; and - Figure 9 showing steps of harvesting culture medium; and

- Figure 10 showing micro sampling steps for analysis. Figures 6 to 10 are described in the following examples.

The following examples are intended to describe the operation of an automatic culture device according to the invention designated "BED". This is a preferred device according to the invention which imposes no limitation to the invention claimed in the present application.

EXAMPLES

The operating plan of a continuous cell culture method according to the invention, in a closed chamber (not shown) uses any culture support, rather of the culture support type each Figure corresponding to a functional diagram explaining specific steps of operating a continuous culture device according to the invention, in a version comprising:

- Four crop supports 11, I2, 13 and 1 ₄ each comprising a single containment tank 2i, 2 ₂ , 2 ₃ and 2 ₄ and a single culture vessel 6'i, 6 ' ₂ , 6' ₃ and 6 ' ₄ . Each culture support is equipped with a bubbler arm 5i, 5 ₂ , 5 ₃ and 5 ₄ , intended to be bonded to a sterile aerating rod (or bubbling nozzle), a stock of ventilation rods 4i. , 4 ₂ , 4 ₃ and 4 ₄ (not shown), and an ejection lock of the culture vessel 18 ₁ , 18 ₂ , 18 ₃ and 18 ₄ ,

It is possible, in a variant, to consider that the references 11, 12, 13 and 14 represent removable block type culture blocks 12 comprising several (for example two, four, six ...) culture vessels. a stock (or stock) of SR sterile culture vessels,

a stock (or stock) of transfer pipettes SPT, a stock (or stock) of dilution pipettes SPD for adding fresh culture medium,

a stock of diluent medium SMCD or sterile culture medium,

- an SE sampling airlock to take samples out of the enclosure

an airlock of pipettes and contaminated material SMPM,

- An airlock of SA analysis station allowing the introduction of equipment or reagents or others to carry out measurements,

a multifunction articulated arm running through the enclosure in the length, the three-dimensional displacement perimeter of the arm being marked by the zone Di ₉ ;

The transfer pipettes generally have a volume approaching the volume of a culture vessel, i.e. most often 20 to 30 ml, and are intended to carry most of the culture medium from a container of culture to another. Dilution pipettes, they generally have a much lower volume, for example most often from 2 to 3 m L.

The arrows and references of the type T _xy , where x is the number of the Figure and y the number of the path for said Figure, indicate the path of the articulated arm at the beginning of the culture (Figure 6), during an operation. dilution method (FIG. 7), during a change operation of a culture vessel (FIG. 8), during a crop sampling operation (FIG. 9), and during an operation for taking a culture sample for analysis inside the closed chamber (FIG. 10).

Functioning of the BED device

Basic operating cycle

1 °) Initial stages of a culture

The operator installs in the enclosure of the device, in the respective storage areas, the following chargers / racks: - Sterile culture vats (or culture vessels),

- Sterile transfer pipettes,

- Sterile dilution pipettes,

- Bubble ducts or aeration rods, sterile, intended to be linked to the bubbler arms 5i, 5 ₂ , 5 ₃ and 5 ₄ ,,

The operator installs in the cell culture device, in the respective storage areas, closed sterile reservoirs containing the different diluents.

The operator opens the tanks containing the different diluents. The operator installs in the robot at the dedicated location the closed sterile culture container containing pure culture No. 1.

The operator opens said culture vessel.

The multi-function automated articulated arm captures a tank on its rack in the tank SR (FIG. 6, path 1 T ₆ i), and transports it to the culture support dedicated to this culture (FIG. 6, path 2 T ₆₂ ). In the case shown in FIG. 6, the first culture support 1 i is filled by the pure culture No. 1 in a culture vessel or container 6 'i, then successively, as explained hereinafter, the other three supports of Culture 1 ₂ , 13 and 1 ₄ are filled with a culture medium, by transfer through a transfer pipette from a culture medium into a corresponding culture vessel or vessel 6 ' ₂ , 6' ₃ and 6 ' ₄ .

The multi-function automated articulated arm pierces the protective film of the culture vessel (sterile vessels).

The multi-function automated articulated arm draws a transfer pipette onto its rack in the SPT reservoir (Figure 6, path 3 T ₆ 3).

The multi-function automated articulated arm, equipped with a transfer pipette, picks up the culture in the tube containing the culture (Figure 6, path 4 T ₆₄ ).

The multi-function automated articulated arm transports the culture to the corresponding support and empties the transfer pipette into the culture vessel (Figure 6, path 5 T ₆ s). The automated robotic arm draws a sterile bubbling cane, then positions the sterile bubbling cane in the culture vessel (Figure 6, path 6 T ₆ 6). The automated articulated bubbler arm opens the gas supply.

2 °) Continuous cultivation

The automatic device receives the trigger signal given by the detector located in the culture medium. This one is programmed to follow a physicochemical parameter and to emit a signal when one reaches a critical value of this parameter.

The automated articulated arm then draws a sterile dilution pipette into the corresponding SPD reservoir (FIG. 7, path 1 T ₇₁ ). The automated articulated arm draws a determined volume of a No. 1 diluent and then draws a sterile air bubble to maintain sterilization during transport (Figure 7, path 2 T ₇₂ ).

The automated articulated arm repeats this operation n times to take the desired volume of n diluents. The multi-function automated articulated arm transports the dilution pipette to the corresponding support and empties the dilution pipette into the culture vessel (Figure 7, path 3 T ₇₃ ).

The multi-function automated articulated arm positions the dilution pipette for evacuation of excess volume. The multi-function automated articulated arm evacuates the surplus volume, two evacuation modes are possible: a) the multi-function automated articulated arm positions the dilution pipette in the culture and takes a determined volume of culture then sucks a bubble of air, or b) the multi-function automated articulated arm positions the dilution pipette at a specific height in the culture vessel (corresponding to to the desired culture volume) and draws all the excess culture volume then sucks an air bubble.

The multi-function automated articulated arm transports the dilution pipette containing the surplus culture to the top of the ejection chamber, which acts as the nearest liquid / waste disposal station (to maintain sterilization) and empties the contents. of the dilution pipette in said ejection chamber 18i, I 82, 183 or 18 ₄ (FIG. 7, path 4 T ₇₄ )

Similarly, the multi-function automated articulated arm transports the empty dilution pipette over the ejection chamber, which acts as the nearest solids / bin disposal station (to maintain sterilization) and ejects the pipette from the container. dilution in said ejection chamber 181, 18 ₂ , 18 ₃ or I 84 (FIG. 7, path 4 T ₇₄ )

3 °) Change of tank

The automatic device detects the trigger signal of a culture container change either at the end of a given time cycle or following the detection of a biofilm.

The multi-function automated articulated arm grabs a tank on its rack in the SR tank and transports it to the transient position dedicated to this culture (Figure 8, paths 1 and 2 T ₈ i and T ₈₂ ).

The automated articulated arm draws a sterile transfer pipette into the SPT reservoir (Figure 8, path 3 T ₈ 3)

The automated articulated bubbler arm closes the gas supply and releases the used bubbling cane from the culture.

The automated bubbler arm ejects the used bubbling nozzle into the ejection chamber which acts as the nearest solids / bin disposal station (to maintain sterilization) and ejects the used cane into said chamber. ejection 181, I 82, 183 or 18 ₄ . The automated articulated arm picks up the culture.

The multi-function automated articulated arm transports the transfer pipette containing the culture to the new tank in the position transient dedicated to this culture and empties the transfer pipette into the new culture vessel (Figure 8, path 4 T ₈₄ ).

The multi-function automated articulated arm transports the empty transfer pipette over the ejection chamber which acts as the nearest solids / bin disposal station (to maintain sterilization) and ejects the transfer pipette into said ejection chamber 18i, I 82,

I 83 or 18 ₄ (FIG. 7, path 4 T ₇₄ ) (FIG. 8, path T ₈₅ ).

The automated articulated bubbler arm draws a sterile bubbling cane (Figure 8, path 6 T ₈₆ ). The automated articulated bubbler arm positions the sterile bubbling cane in the culture vessel.

The automated articulated arm bubbler opens the gas supply

The culture vessel is ejected via the ejection lock (Figure 8, path 7 T ₈₇ ).

4 °) Selection of microorganisms growing in suspension:

The cultures are made in a culture vessel (disposable plastic tanks) in a culture support as shown in FIG.

This disposable culture container can be regularly replaced, when necessary (biofilm development) by a new sterile container via the action of a robotic arm, as described above.

An overview of the culture device is for example given in FIG. 5.

During a tank change, the arm ensures the pumping of the medium in a sterile pipette during the exchange of the culture tanks, then places the culture medium in the new sterile tank (exchange function of the container). This robotic arm also ensures the pipetting functions of a portion of the culture and the replacement of the volume taken up by the same volume of fresh medium (dilution function), the dilution frequency can be fixed by the experimenter (chemostat) or driven by the cell density of the culture (turbidostat).

Any physical object coming into contact with the culture (aeration tube, transfer pipette, dilution cones) is replaced after each use by a new sterile object.

The container (= the culture tank) is placed in a thermostated metal case to keep the culture at a controlled temperature. This case is equipped with one or more forks (transmitter / receiver at different wavelengths) to measure the cell density (visible, IR) in the tank, as well as the concentration of some absorbing molecules.

The culture vessel and the thermostated case are located in a confinement tank inside which a sterile gaseous flow, for example sterile air, flows continuously around the case and the tank. to achieve a constant sterile containment of the latter (see Figure 1).

In this way, only the organisms that develop in suspensive mode (and therefore subject to dilution) are maintained in long-term growth under selective conditions.

Due to the use of single-use disposable tubs and accessories and the absence of a complex fluidic network, the system does not require complex and time-consuming sterilization operations.

All tanks in place of the system are functional and ensure the development of selective cultures.

In addition, the system makes it possible to control the culture either with constant degree of dilution and with constant volume with compensation of the evaporation, either with variable degree of dilution without compensation of the evaporation, or still with degree of variable dilution with compensation of evaporation.

The possibility of finely controlled liquid phase molecular contributions via the action of the robotic arm makes it possible to envisage the precise modulation of the selection pressures applied to the cultures for a large number of constituents without requiring the multiplication of dedicated branches of the fluidic network. Moreover, the addition of difficult to handle products (such as acetaldehyde which is volatile and reactive) is possible without having to pass this product through a complex fluid network.

In addition, the system makes it possible to envisage the addition of solid particles such as cells immobilized in alginate beads, water-immiscible liquids and various additives that are virtually impossible to convey in the aqueous phase in a complex fluidic network.

In addition, small volumes of aliquots can be collected via the robotic arm in a sterile manner at any point in the culture to be transferred to any type of analytical equipment.

(HPLC, PCR, ELISA, MS ...) external to the BED system and whose results can be integrated for the control of the current crop.

In addition, the system makes it possible to use different forms of culture vats according to the needs of the culture requirements of the microorganisms.

5 °) Selection of microorganisms developing in the form of biofilms

The device described above allows the addition of plates of different materials in the culture vessel via the robotic arm for the selection of cells developing in the form of biofilms.

The fraction of the culture which, fixed on these plates, can be preserved while the culture medium containing the cells in suspension removed and replaced by fresh medium. Similarly, plates covered with biofilms can be moved using the robotic arm into a new sterile culture vessel.

In this way, it is possible to select cell variants that develop the ability to attach to a support or to aggregate to form structures such as biofilms.

Optional additional actions

1 °) Adding a reagent All types of reagents can be considered in this context for example chemicals biochemical products (proteins, DNA etc.), biological (cell suspensions) etc.

The robot detects the trigger signal of adding a reagent. The automated articulated arm takes a micropipette of sterile reagent.

The automated articulated arm transports the micropipette of sterile reagent to the reservoir containing the desired reagent.

The automated articulated arm takes up a negligible volume in front of the total volume of determined culture of the reagent (for example 25 μL of reagent) then takes a bubble of sterile air.

The multi-function automated articulated arm transports the micropipette containing the reagent to the corresponding incubator and empties the dilution pipette into the culture vessel. The multi-function automated articulated arm rinses the wall of the micropipette through a cycle of aspirations. repressions in the micropipette

The multi-function automated articulated arm transports the reagent micropipette over the solid element / waste disposal station and ejects the reagent micropipette into the solid element evacuation station

2 °) Routine sample collection

The operator installs a sterile tube in the sampling station or SE aliquot.

The operator opens the sterile tube in the sampling station or aliquot SE.

The robot detects the trigger signal of a dilution and the trigger signal of a sample. The robot performs a dilution as described in the previous section in paragraph 2) with one step: the automated articulated multi-function arm carries the dilution pipette containing the surplus culture above the SE station and depositing the contents of the dilution pipette into the open sterile tube (Figure 9, path 3 T ₉₄ ).

The operator rebases the sterile tube in the SE station. The operator closes the sampling tube and retrieves the culture medium via the sampling chamber SE.

3 °) Emergency sample collection The operator installs a sterile tube in the sampling station

SE.

The operator opens the sterile tube in the sampling station SE.

The robot detects the trigger signal of a sample. The automated articulated arm draws a sterile dilution pipette into the SPD reservoir (Figure 9, path 1 T ₉₁ ).

The automated articulated arm transports the sterile dilution pipette to the culture in question (FIG. 9, path 2 T ₉₂ ).

The automated articulated arm takes a determined volume (for example 2 mL) of culture and then an air bubble

The multi-function automated articulated arm transports the dilution pipette containing the culture sample over the sampling station and deposits the contents of the dilution pipette into the open sterile tube (Figure 9, path 3 T ₉₃ ). The multi-function automated articulated arm transports the used dilution pipette over the solid waste / waste disposal station and ejects the dilution pipette into the solid element discharge station (Figure 9, path 4 T ₉₄ ).

The operator rebases the sterile tube into the sampling station.

The operator closes the sampling tube and retrieves the aliquot of the culture. 4 °) Collection of micro sample for analysis

The robot detects the trigger signal of a micro sample sample for analysis. The automated articulated arm draws a sterile micropipette into the SEPD reservoir (Figure 10, path 1 T101).

The automated articulated arm transports the sterile micropipette to the culture in question (Figure 10, path 2 Tw ₂ ).

The automated articulated arm takes a determined micro volume (for example 10 μL) of culture and then an air bubble.

The multi-function automated articulated arm transports the micropipette containing the culture sample above the analysis station SE and deposits the contents of the micropipette in the position assigned to this sample in the analytical platform (Figure 10, path 3 T103 ). The multi-function automated articulated arm transports the used micropipette over the pipette ejection port and contaminated material SEPM acting as a solid waste / bin disposal station and ejects the micropipette into said SEPM chamber (FIG. journey 4 Ti 04) -

Claims

1. A continuous cell culture method for the selection of static cell variants or proliferation in suspension, characterized in that it comprises the following steps: a) is seeded with the aid of one or more living cells a culture medium liquid contained in a first culture vessel kept open in a closed chamber, b) said cells are brought into said culture medium at a determined growth stage, corresponding to a given cell density or to a physico-chemical parameter measured in the culture medium, c) the cell density in the culture medium, or the value of said physico-chemical parameter, reached in step b), is kept substantially constant by adding fresh culture medium or a diluent to the culture medium; said culture vessel, d) pipetting part of the culture medium of the culture obtained in c) containing the cells in suspension so as to maintain (e) transferring a fraction of the culture medium obtained in d) into a second culture vessel, f) removing said first culture vessel with the remaining culture fraction contained therein; g) after several generations of culture in the second vessel, the suspension-proliferating cells and / or the static cell variants are selected.

2. A method of continuous cell culture for the selection of cell variants proliferating in suspension, characterized in that it comprises the following steps: a) a liquid culture medium contained in a first culture vessel kept open in a closed chamber is seeded with the aid of one or more living cells; b) the cells are introduced into the culture medium at a stage of determined growth, corresponding to a given cell density or to a physicochemical parameter measured in the culture medium, c) the cell density of the culture, or the value of said physico-chemical parameter, is maintained substantially constant at the stage b) by adding fresh culture medium or at least one diluent to said culture vessel, d) pipetting a portion of the culture medium containing the cells in suspension to maintain the volume of the culture e) transferring a fraction of the culture obtained in d) in which the cells are suspended, in a second culture vessel replacing the first; f) removing said first culture vessel with the remaining culture fraction contained therein; g) after several generations of culture in the second container, the cells proliferating in suspension are selected.

3. Continuous cell culture method for the selection of static cell variants, characterized in that it comprises the following steps: a) one inoculation with the aid of one or more living cells a liquid culture medium contained in a first culture vessel in which is placed at least one solid surface, preferably a plate, said container being kept open in a closed chamber, b) the cells are brought into the seeded culture medium at a determined growth stage, corresponding to a given cell density or to a physicochemical parameter measured in the culture medium, c) the cell density of the culture, or the value of said physico-chemical parameter, reached in step b), is kept substantially constant by adding fresh culture medium or at least one diluent to said culture vessel, d) pipetting part of the culture medium obtained in c) containing the cells in suspension so as to maintain the volume of the culture, e) transferring said solid surface on which a fraction of the culture obtained in d) s is deposited in a second culture vessel replacing the first, f) said first culture vessel is removed with the remaining culture fraction it contains, g) the static cell variants fixed on said culture are selected after several generations of culture. solid surface.

4. Method according to claim 3, characterized in that said solid surface is made of a treated material to prevent adhesion of the cells.

5. Method according to any one of claims 1 to 4, characterized in that said culture vessels are kept open at their apex, and that a gaseous flow, such as sterile air, is applied continuously, on the outskirts of their opening.

6. Process according to any one of claims 1 to 5, characterized in that a sterile gaseous flow is applied from the top to the bottom of the culture vessels.

7. Method according to the preceding claim, characterized in that maintains the sterile gaseous flow from top to bottom by creating a depression at the periphery of the culture vessel.

8. Process according to claim 7, characterized in that the sterile gaseous stream is discharged from the base enclosure of said culture vessels.

9. Method according to any one of claims 1 to 8, characterized in that at least the different steps a) to f) are performed in a closed chamber.

10. Method according to any one of claims 1 to 9, characterized in that repeats the steps a) to f) one or more times before proceeding to step g).

11. A method according to any one of claims 1 to 10, characterized in that the maintenance of a substantially constant cell density in the culture in step c) is carried out by dilution of the culture with fresh medium. maintaining a constant volume of culture medium in the culture vessel.

12. Method according to any one of claims 1 to 11, characterized in that the transfer of the culture medium containing the cells in suspension in the second culture vessel is operated by pipetting with a sterile pipette.

13. Method according to any one of claims 1 to 12, characterized in that the first culture container used, is evacuated with the aid of an airlock outside the closed chamber.

14. A method according to any one of claims 1 to 13, characterized in that the culture vessel in operation is placed in a thermostated cell for regulating the temperature of the cell culture.

15. Method according to any one of claims 1 to 14, characterized in that several cultures are carried out simultaneously in several culture media placed in the same enclosure.

16. Method according to any one of claims 1 to 15, characterized in that a sampling of the culture is performed regularly.

17. Method according to any one of claims 1 to 16, characterized in that the culture media or the culture vessels are disposable.

18. A method according to any one of claims 1 to 17, characterized in that gas is injected into the cells in culture by means of a bubbling device introduced into the culture vessel.

19. Method according to any one of claims 1 to 18, characterized in that several process steps are performed using one or more automated arms allowing movement within the enclosure.

20. Process according to any one of claims 1 to 19, characterized in that the cells are cultured continuously over a number of generations greater than 10 ² , preferably greater than 10 ⁴ , more preferably greater than 10 ⁶ , and still more preferably greater than ¹⁰ generations, without opening the chamber on the outside environment.

21. Method according to any one of claims 1 to 20, characterized in that it is applied to the culture of autotrophic microorganisms and said culture is illuminated during the various steps of said method.

22. Culture support (18) for performing an open continuous cell culture, characterized in that it comprises: at least one culture vessel (1) open at its top (6) capable of containing a medium of liquid culture; at least one upwardly open containment tank (2) in which said culture vessel (1) is housed; a space (7) between said culture vessel (1) and said containment tank (2), able to circulate a gas flow at the periphery of the container opening, from top to bottom, between the culture vessel (1) and the inner walls of the containment tank (2); and extraction means (8) of said gas stream located in the lower part of the containment tank (2).

23. Culture support according to claim 22, characterized in that said means for extracting the gas flow consists of one or more orifices (8) passing through the gas stream.

24. culture support according to claim 22 or 23, characterized in that it is in the form of a removable block (12) in which several of said containment bins are grouped and in which are housed at least one of said containers of culture (1).

25. culture support according to one of claims 22 to 24, characterized in that said lower portion of the containment tank (2) is flush against a base provided with complementary means (14) for extracting said gaseous flow flowing between the culture vessels (1) and the walls of the containment tanks (2), such as a pipe connected to a vacuum pump, or one or more gas distribution means.

26. Culture support according to one of claims 22 to 25, characterized in that it comprises, in addition, at least one means for regulating the temperature (3) of the internal volume of said containment tank (2).

27. Support according to claim 26, characterized in that said means for regulating the temperature of the internal volume (7) of said containment tank consists of a heating resistor included in at least one of the walls of said containment tank (2). .

28. Culture support according to any one of claims 22 to 27, characterized in that it further comprises at least one air injection means in the medium (s) of culture, contained (s) in the said container (s), such as one or more shank (s) (4).

29. Culture support according to any one of the claims

22 to 28, characterized in that it further comprises at least one means for optical measurement (9) of the density of the cells present in the culture medium contained in the culture vessel (1).

30. Culture medium according to any one of claims 22 to 29, characterized in that it comprises, in addition, at least one light generating means (15) for the cultivation of microorganisms in autotrophic mode, located in the space between the culture vessel and the inner walls of said containment tank (2), or included in one of the walls of said containment tank.

31. Cell culture device for continuous growth of cells in open mode, characterized in that it comprises: - an enclosure (17); means for generating a sterile gaseous flow (23) passing through said enclosure; one or more culture media (18) according to any one of claims 22 to 30, positioned in said enclosure (17), for performing an open cell culture.

32. Cell culture device according to claim 31, characterized in that it further comprises: at least one means of renewal of the culture medium placed inside said enclosure.

33. Cell culture device according to claim 31 or 32, characterized in that it further comprises: at least one culture vessel (1) adapted to replace that contained in the culture medium (18).

34. Device according to any one of claims 31 to 33, characterized in that the means for generating the sterile gas stream (23) is placed in the upper part of the enclosure (17) so that said gas stream sterile goes from the top to the bottom of the culture support (18).

35. Cell culture device according to any one of claims 31 to 34, characterized in that it further comprises at least one means for extracting the sterile gaseous flow (8) at the base of said culture vessel.

36. Device according to claim 35, characterized in that the means for extracting the sterile gas stream (8) creates a vacuum in the space (7) located between the culture vessel (1) and the containment tank (2). ) of the culture support (18).

37. Device according to claim 35 or 36, characterized in that said extraction means (8) is associated with at least one suction means of the air adapted to suck the air surrounding the periphery of the opening (6) of the culture vessel.

38. Device according to any one of claims 32 to 37, characterized in that the means of renewal of the culture medium comprises means for transferring (20) a portion of the contents of the culture vessel to an evacuation zone. .

39. Device according to any one of claims 32 to 38, characterized in that the means of renewal of the culture medium comprises means for transferring (20) fresh medium from a reserve located inside the enclosure ( 17) to the culture vessel.

40. Device according to one of claims 38 or 39, characterized in that the transfer means comprises a pipette (20) and a suction means fresh medium or used in said pipette.

41. Device according to one of claims 31 to 40, characterized in that it comprises, in addition - at least one outlet means to the outside of the enclosure of the transfer means (20) and / or at least one culture vessel.

42. Device according to claim 41, characterized in that said output means comprises an airlock.

43. Device according to any one of claims 31 to 42, characterized in that it comprises at least one air injection means (4, 21) in the culture.

44. Device according to any one of claims 31 to 43, characterized in that it comprises at least one automated arm (19) adapted to make displacements within the enclosure.

45. Device according to any one of claims 31 to 44, characterized in that the enclosure (17) is closed.

46. Device according to any one of claims 31 to 45, characterized in that it comprises a plurality of culture media positioned in said chamber, to perform in parallel several open cell cultures.

47. Device according to any one of claims 30 to 46, characterized in that it allows the implementation of the method according to any one of claims 1 to 21.