Method for supplying a bioreactor with culture medium, and an associated system

Abstract

The invention relates to a method for supplying a bioreactor with culture medium, comprising: providing a liquid matrix source; providing a liquid flow line between the source of the liquid matrix and the bioreactor; initiating a flow of the liquid matrix through the liquid flow line; providing at least one source of enrichment substances; injecting at least one nutrient into the flow of the liquid matrix in order to form a culture medium with a defined concentration of enrichment substances in the liquid flow line; and providing a desired quantity of the culture medium to the bioreactor. The invention also relates to an associated system.

Description

METHOD FOR FEEDING A BIOREACTOR AND AN ASSOCIATED SYSTEM IN A CULTURE MEDIUM TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a method of feeding a bioreactor into a culture medium and an associated system, in particular for experimental cultures of aquatic organisms (e.g. unicellular microorganisms, such as microalgae, fungi, bacteria, yeasts, protists or multicellular microorganisms such as gametophytes, mosses, larvae, fish, molluscs ...) where the need for a controlled sterile input in enrichment of substances (e.g. nutrients, pollutants or even drugs) and in flow rate is necessary (e.g. continuous culture mode of chemostats, turbidostats). TECHNICAL BACKGROUND

[0002] To develop experimental cultures, such as microorganisms in bioreactors, a culture medium is required. This culture medium can be a liquid matrix enriched with one or more nutrients (or pollutants, or drugs, for example, in the case of chronic exposure of the culture medium or microorganisms). For instance, in microalgae cultivation, a culture medium might consist of a seawater matrix enriched with nutrients such as nitrogen, phosphorus, glucose, minerals, metals, and vitamins.To determine, through experimentation, which nutrients and their concentrations are best for growing a culture or to study ecophysiology in relation to nutrition in a bioreactor, nutrient combinations and their concentrations can be varied over time or in several bioreactors operating in parallel, observing the effect of the input(s) on the growth and development of the resulting cultures. It is therefore necessary to provide precisely defined quantities of culture media containing specific concentrations of nutrients and pollutants. or desired drugs (for example, in the case of chronic exposure). In some applications, sterile conditions of the input and / or the bioreactor are necessary (for example, when studying an aquatic organism under axenic conditions). In these applications, it is therefore important that the culture medium be sterile to avoid any contamination that could invalidate the experiments.

[0003] Up to now, culture medium has typically been supplied to a bioreactor 10 undergoing continuous growth experimentation using a system as illustrated in Figure 1. Culture medium 42 is supplied as needed from a reservoir 20 by actuation of a pump, for example, a peristaltic pump 30 (other types of pumps are sometimes implemented, such as diaphragm pumps), with the culture medium flowing through a flow line 40 to a sterile inlet of the bioreactor. The culture medium 42 is prepared in batches. For example, culture medium is prepared in reservoir 20 by diluting a certain concentration of nutrient medium with water. When a reservoir 20 is empty or when a new type or concentration of enrichment is required, reservoir 20 is manually replaced with another batch.

[0004] The sterile culture medium is therefore prepared in batches by the experimenter. For example, the experimenter mixes concentrated nutrient solution and seawater directly in the reservoir. The reservoir can be a bottle, a barrel, or a bag. This introduces several risks to the maintenance and reliability of the experiments. Such a conventional system presents risks of errors in the enrichment dosage during preparation, aging of the stored medium, and breach of system sterility during preparation. Other risks of error and breach of sterility arise from repeated connections of the new batch to the bioreactor's feed system, and from flow rate drift (and therefore control of the microorganism growth rate) due to wear on the peristaltic pump tubing.In addition, batch preparation of culture media over prolonged periods creates an experimental burden that considerably hinders replication, or even the ambition of experiments, and more broadly the diffusion of continuous culture in laboratories.

[0005] Several methods for automating the process have been proposed, as illustrated in Figure 2. Here, the culture medium 42 is supplied to the bioreactor 10 from a reservoir 20 under the automated control of a controller 50. Aqueous matrix fluidic reservoirs 60 and concentrated nutrient solutions 62 are connected to the reservoir 20, and their input is controlled by the controller 50, so that the culture medium of a desired nutrient concentration is mixed, on demand, in the reservoir 20. The culture medium mixture is prepared on demand by filling the reservoir 20 in response to signals from the level sensors 12 and 14 on the reservoir 20. Filling is triggered by a signal from the low-level sensor 12, and the amount of concentrated nutrient solution 62 to be mixed with the aqueous matrix fluid 60 is determined by the volume of the reservoir 20 at the high-level sensor 14.The resulting culture medium is fed into bioreactor 10 through the sterile inlet.

[0006] Optionally, a flow meter 70 can be incorporated into the flow line 40, and the operation of the peristaltic pump 30 can thus be controlled according to the measured flow rate to take into account drift due to wear of the tubes.

[0007] This solution addresses part of the main technical problem (manual preparation) through automation. However, it still requires sequential preparation of the culture medium and carries the risk of solution residue within the system. Furthermore, sterilizing the entire fluidic system, including the flow meter, is difficult (for example, by autoclaving). Until now, flow meters used in such systems—for instance, for laboratory flow rates—have been integrated directly into the liquid flow line. Their integration into the system therefore poses a risk of compromising sterility, as they cannot be sterilized by autoclaving.In order to sterilize the system, it would therefore be necessary to remove the flow meter - which itself presents a risk of sterility to the rest of the system due to the disconnection - and to thoroughly clean the flow path through the flow meter.

[0008] It would therefore be advantageous to provide a method for feeding culture media into a bioreactor and an associated system that mitigates the problems mentioned. SUMMARY OF THE INVENTION

[0009] The present invention relates to a method for feeding culture medium into a bioreactor, comprising: the provision of a liquid matrix source; the provision of a liquid flow line between the liquid matrix source and the bioreactor; the initiation of a liquid matrix flow through the liquid flow line; the measurement of the liquid matrix flow through the liquid flow line by means of a non-invasive flowmeter comprising a conduit of constant cross-section, a bubble injector adapted to inject one or more sterile bubbles into the conduit, a first optical sensor in a first position on the conduit downstream of the bubble injector and disposed to detect the passage of said one or more bubbles, and a second optical sensor in a second position on the conduit downstream of the first optical sensor and disposed to detect the passage of said one or more bubbles;in which the flow rate is calculated based on a time difference between the second optical sensor and the first optical sensor respectively detecting the passage of said one or more bubbles; the provision of at least one source of enrichment substances; the injection of at least one enrichment substance into the flow of the liquid matrix to form a culture medium of defined concentration of enrichment substances in the liquid flow line; and the provision of a desired quantity of the culture medium to the bioreactor.

[0010] For example, the enrichment substance can be one or more nutrients, one or more pollutants, one or more drugs, or any other substance.

[0011] According to one embodiment of the present invention, the conduit is a portion of the liquid flow line.

[0012] According to one embodiment of the present invention, the flow meter is external to the conduit and the flow path through the flow meter therefore includes the conduit itself. Thus, the flow meter can easily be removed from the pipe so that the latter can be sterilized.

[0013] According to one embodiment of the invention, the bubble(s) comprise a bubble front. According to another embodiment of the invention, the bubble(s) comprise a sterile gas.

[0014] According to one embodiment of the invention, the detectors are optical sensors. According to one embodiment of the invention, each of the optical sensors comprises a light source and an associated light detector.

[0015] According to one embodiment of the invention, the conduit has a constant cross-section at least between the first detector and the second detector.

[0016] Advantageously, the process according to the invention allows for the preparation of a nutrient medium (or enriched medium) with a variable and / or time-controlled composition, therefore dynamic, and in a sterile manner, since the enriching substance(s) are injected directly into the flow line, in real time and continuously. Such a process makes it possible to limit sources of residual contamination.

[0017] According to one embodiment of the invention, the liquid matrix is ​​an aqueous matrix. Preferably, the aqueous matrix comprises seawater, such as that used in culture media for microalgae cultures. Of course, it is perfectly possible to use fresh water within the scope of the present invention.

[0018] According to one embodiment of the invention, said at least one source of enrichment substances is in the form of at least one syringe pump in sterile communication with the liquid delivery line. For example, the syringe pump can be filled once or refilled via its sterile connection to a concentrated solution of enrichment substances (for example, in a bottle). A syringe pump is a practical means of maintaining sterility by forming a connection with the delivery line. Furthermore, it allows for very precise dosing.

[0019] According to one embodiment of the invention, said at least one syringe pump is removably connected to the liquid flow line. In this way, one can The syringe pump(s) can be quickly, easily, and sterilely mounted and dismounted on the flow line. According to another embodiment of the invention, the syringe pump(s) is / are connected to the liquid flow line so that they are sterilized along with the line, thus maintaining sterility. Indeed, during mounting / dismounting, there is a risk of compromising sterility.

[0020] According to one embodiment of the invention, initiating the flow of the liquid matrix through the liquid flow line involves actuation of an associated pump. In one embodiment of the invention, the pump is a peristaltic pump to ensure a sterile line. Conversely, gravity-fed systems, for example, could be considered.

[0021] According to one embodiment of the invention, the method further comprises measuring, by the flow meter, the flow rate of the liquid matrix through the liquid flow line. In another embodiment of the invention, said at least one enriching substance is injected at a flow rate that is a function of the liquid flow rate measured by the flow meter. In this way, the concentration of the enriching substance(s) in the liquid can be precisely mixed.

[0022] According to one embodiment of the invention, supplying a desired quantity of the culture medium to said bioreactor includes actuation of a pump between the liquid flow line and the bioreactor.

[0023] According to one embodiment of the invention, the process is a method for supplying a culture medium to a plurality of bioreactors (210a-c) and the supply of a desired quantity of the culture medium includes a preliminary step of initiating at least one flow of the liquid matrix by means of actuation of a set of valves between the liquid flow line and the plurality of bioreactors.

[0024] According to one embodiment of the invention, the steps of initiating a flow of the liquid matrix, injecting at least one enrichment substance, and supplying a desired quantity of the culture medium are carried out in an automated manner.

[0025] According to one embodiment of the invention, a culture medium having a dynamically adjustable concentration of enriching substances is supplied to the bioreactor. A culture medium having an instantaneously adjustable concentration of enriching substances can be supplied to the bioreactor(s).

[0026] Another embodiment of the invention relates to a system for supplying enrichment substances to feed the culture medium of a bioreactor. Naturally, the feeding system can supply several bioreactors. More specifically, the system comprises: a liquid matrix source; a liquid flow line between the liquid matrix source and the bioreactor and means for making the liquid matrix flow through the liquid flow line; at least one enrichment substance source in a sterile connection with the liquid flow line; and means for injecting said at least one enrichment substance source into the flow line to form a culture medium of defined concentration of enrichment substances in said flow line.and means for supplying a desired quantity of the culture medium to the bioreactor, a non-invasive flowmeter comprising a conduit of constant cross-section, a bubble injector adapted to inject one or more sterile bubbles into the conduit, a first optical sensor in a first position on the conduit downstream of the bubble injector and disposed to detect the passage of said one or more bubbles, and a second optical sensor in a second position on the conduit downstream of the first optical sensor and disposed to detect the passage of said one or more bubbles; wherein the flow rate is calculated as a function of a time difference between the second optical sensor and the first optical sensor respectively detecting the passage of said one or more bubbles, the flowmeter being configured to measure a flow rate of the liquid matrix through the liquid flow line.

[0027] According to one embodiment of the present invention, the conduit is a portion of the liquid flow line.

[0028] According to one embodiment of the present invention, the flow meter is external to the conduit, and the flow path through the flow meter therefore includes the conduit itself. Thus, the flow meter can easily be removed from the conduit so that the latter can be sterilized.

[0029] According to one embodiment of the invention, the bubble(s) comprise a bubble front. According to another embodiment of the invention, the bubble(s) comprise a sterile gas.

[0030] According to one embodiment of the invention, the detectors are optical sensors. According to one embodiment of the invention, each of the optical sensors comprises a light source and an associated light detector.

[0031] According to one embodiment of the invention, the conduit has a constant cross-section at least between the first detector and the second detector.

[0032] According to one embodiment of the invention, the liquid flow line is part of a liquid circuit which is a single unit and which can be completely disassembled from the rest of the system.

[0033] According to one embodiment of the invention, the means for making the liquid matrix flow through the liquid flow line include a pump (170), which is preferably a peristaltic pump. Conversely, gravity-fed systems can be considered, for example.

[0034] Advantageously, such a system allows the preparation of nutrient medium with a precise and time-varying composition, therefore dynamic, and in a sterile manner, because the nutrient(s) are injected directly into the flow line, in real time and continuously, and as the flow meter is non-invasive, it has no negative impact on the sterility of the conduit and therefore of the system.

[0035] According to one embodiment of the invention, the liquid flow line is part of a single-piece liquid circuit that can be completely detached from the rest of the system. In this way, the circuit can be separated from the rest of the system (the liquid matrix source, the means for making the liquid matrix flow through it). the liquid flow line, nutrient sources, etc., which are all non-invasive) without further handling thereafter, to be fully processed in an autoclave to ensure optimal sterility, significantly reducing the risk of breaking sterility over time.

[0036] According to one embodiment of the invention, the liquid matrix source preferably comprises a reservoir containing an aqueous liquid. In another embodiment of the invention, the liquid matrix source is connected to a water distribution network.

[0037] According to one embodiment of the invention, said at least one source of enrichment substances comprises at least one syringe pump containing a respective enrichment substance and in sterile communication with the liquid flow line, preferably removably connectable to the liquid flow line via a sterile interconnection. A syringe pump is a practical means of maintaining sterility by forming a connection with the flow line. Indeed, the syringe pump and the reservoir(s) of concentrated nutrients can be autoclaved together with the main circuit. Furthermore, the syringe pump allows for very precise dosing. According to one embodiment of the invention, a filter can be placed downstream of the syringe pump. According to another embodiment of the invention, any solution that allows for sterile refilling of the syringe pump can also be implemented.

[0038] According to one embodiment of the invention, the means for making the liquid matrix flow through the liquid flow line include a pump.

[0039] According to one embodiment of the invention, the flow meter is configured to measure a flow rate of the liquid matrix through the liquid flow line.

[0040] According to one embodiment of the invention, the means for initiating the supply of the culture medium to the bioreactor include a valve between the liquid flow line and the bioreactor.

[0041] According to one embodiment of F invention, the system comprises a plurality of bioreactors with multiple respective valves directing the culture medium to them.

[0042] According to one embodiment of the invention, the system further comprises a controller operationally connected to each of the means for bringing the liquid matrix to flow through the liquid flow line, the means for injecting said at least one source of enrichment substances, and the means for supplying a desired quantity of the culture medium to the bioreactor, so that a culture medium having a dynamically adjustable concentration of enrichment substances can be supplied to the bioreactor in an automated manner. LIST OF FIGURES

[0043] Embodiments of the invention will now be described by way of example only with reference to the drawings in Figures 1 to 5.

[0044] Figure 1 illustrates a known system for manually feeding a bioreactor into the culture medium;

[0045] Figure 2 illustrates another known system for feeding a culture medium to a bioreactor, but in an automated manner;

[0046] Figure 3 illustrates a system for feeding a bioreactor into a culture medium, according to an embodiment with a flow meter upstream of a pump;

[0047] Figure 4 corresponds to Figure 3 and illustrates a system according to a second embodiment with a flow meter downstream of the pump;

[0048] Figure 5a illustrates a flow meter according to one embodiment, with a bubble in an upstream position, near a first optical detector;

[0049] Figure 5b illustrates the flow meter, with the bubble in the downstream position, near a second optical detector;

[0050] Figure 6 illustrates a system for feeding a culture medium to a plurality of bioreactors, according to one embodiment;

[0051] Figure 7 illustrates a comparison between the flow rates measured by a known flow meter and that of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0052] To avoid any doubt, all the characteristics described herein also apply to every aspect of the invention.

[0053] Within the scope of this application, it is expressly provided that the various aspects, embodiments, examples, and alternatives set forth in the preceding paragraphs and / or in the following description and drawings, and in particular their individual characteristics, may be considered independently or in any combination. In other words, all embodiments and / or characteristics of any embodiment may be combined in any manner, unless such characteristics are incompatible.

[0054] To avoid any ambiguity, the terms "may," "and / or," "for example," and any other similar terms used in this document shall be interpreted as non-limiting, such that not every feature thus described is necessarily present. Indeed, any combination of optional features is expressly envisaged without departing from the scope of the invention.

[0055] Also, a person skilled in the art will appreciate that several variations of the described embodiments are conceivable without departing from the scope of the invention.

[0056] We now refer to figures 3 to 5.

[0057] Figure 3 illustrates a first embodiment of a system 100 for supplying enrichment substances to feed the culture medium 142 of a bioreactor 110. More specifically, the system 100 comprises: a liquid matrix source 160, which may be a reservoir or a direct source, such as a freshwater or marine water pipe; and a liquid flow line 140 between the liquid matrix source 160 and the bioreactor 110 and means for bringing the liquid matrix to flow through the liquid flow line 140, these means preferably in the form of a peristaltic pump 130.

[0058] For example, the liquid matrix is ​​an aqueous matrix. For example, it could be seawater.

[0059] For the purposes of this description and by way of non-limiting example, the enrichment substance includes nutrients. Of course, it may also include pollutants or drugs.

[0060] Furthermore, the system 100 includes at least one nutrient source in a sterile connection (or sterile fluidic connection) to the liquid flow line 140, and means for injecting said nutrient source into the flow line to form a culture medium 142 of defined nutrient concentration in the flow line. Preferably, a syringe pump 180 serves both as a nutrient source (preferably, a reservoir is provided upstream to allow refilling of the syringe pump) and as a means for injecting them into the liquid flow line 140 for each nutrient. As illustrated, the injection point is upstream of the pump 130, but it could alternatively be downstream if only one bioreactor is used. A syringe pump allows for precise flow rates and can be mounted and dismounted on the flow line 140 quickly, easily, and asterilely, for example, via a Luer lock connection.A flow meter 170 is configured to measure the flow rate of the liquid matrix through the liquid flow line 140. For example, the flow meter can also regulate the flow rate by implementing a closed loop on the pump. An equivalent scheme can be implemented for the syringe pump.

[0061] Figure 4 illustrates an alternative 100' system, in which all elements are identical and receive the same reference number, supplemented by a ' but in which the 170' flow meter is downstream of the 130' pump. Such a configuration is This is advantageous in the case of a single bioreactor. Furthermore, in this embodiment, the system includes a downstream sterilization filter 190, before the inlet to the bioreactor 110'. The filter 190 can have a 0.2 µm mesh size to filter upstream bacterial growth.

[0062] During operation, as the liquid matrix flows through the liquid flow line 140, the syringe pump(s) 180 are actuated to inject nutrients at a precise flow rate, thus forming a culture medium 142 with a mixture of the desired nutrient concentration. Preferably, the nutrients are injected at a rate that is a function of the measured liquid flow rate. In this way, the nutrients can be precisely mixed to achieve the target concentration. The mixed culture medium 142 can then be directed to supply the desired quantity of culture medium to the bioreactor 110. This feeding can be initiated by activating a purge valve between the liquid flow line 140 and the bioreactor 110.

[0063] While the process can be performed manually, the system preferably includes a controller 150 to automate it. The controller 150 is operationally connected to each of the following: the pump 130; the syringe pump(s) 180; and a flow meter 170 between the liquid flow line 140 and the bioreactor 110. Thus, a culture medium 142 with a dynamically adjustable nutrient concentration can be supplied to the bioreactor 110 in an automated manner.

[0064] As also illustrated with reference to Figures 5a and 5b, the flowmeter 170 is in the form of a bubble injector 172 adapted to inject one or more bubbles 173 into the conduit 140, in conjunction with a first detector 174 and a second detector 176, said detectors arranged to detect the passage of one or more bubbles. The injector can inject a single bubble at a time. It can also inject several bubbles, for example in the form of a bubble train. The bubbles can contain sterile gas, which could be nitrogen. Of course, the sterile gas can also be air or even a noble gas.

[0065] The first detector 174 is in a first position on the flow line 140, downstream of the bubble injector 172. The second detector 176 is in a second position on the flow line, at a distance 'd' downstream of the first detector. Preferably, the detectors are optical sensors, each comprising a light source 174a, 176a and a light detector 174b, associated 176b, which produce a respective signal when a bubble passes and interrupts the passage of light.

[0066] The flow rate through the conduit is calculated based on the time difference between the second and first detectors, which respectively detect the passage of one or more bubbles. The volume of liquid between the two detectors is constant, and the flow rate can therefore be calculated simply as a function of time. If the flow line has a constant cross-sectional area at least between the first and second detectors, the volume of liquid between the two detectors is simply this cross-sectional area multiplied by d.

[0067] The flow line is not interrupted by detectors 174 and 176, and the flowmeter is therefore non-invasive. The liquid (or fluid) flow line is part of a single, continuous liquid circuit that can be completely disconnected from the rest of the system. Specifically, the flow line can be disconnected from the reservoir and bioreactor connections, as well as from the connections to the respective syringe pumps. The experimenter may wish to sterilize the reservoir along with the flow line. The flow line can also be easily disconnected from the first and second detectors and the bubble injector, and from the peristaltic pump. Thus, the liquid circuit can be removed for sterilization.

[0068] As illustrated in Figure 7, the flow rate measurement (Fb) by the non-invasive flowmeter 170 of the invention was experimentally compared to that of a known commercial (invasive) flowmeter, used here as a reference (Fr). Both flowmeters were placed on the same fluid circuit. Data were acquired at four different pump speed settings. As can be seen, a very close correlation was obtained.

[0069] During operation, the speed of the aqueous matrix pump 130 is controlled by a closed-loop control system connected to the flow meter 170, ensuring reliable flow. This non-invasive flow meter, by its very design, maintains the sterility of the system. A significant advantage arises from the use of an optical flow meter. This is based on the principle of discrete injections of sterile gas bubbles into the fluidic section. The passage of two bubble fronts in front of Optical sensors with a known spacing distance allow us to deduce the velocity of the liquid and therefore the flow rate.

[0070] Because the fluid circuit is a single unit, it can be entirely autoclaved to ensure optimal sterility. The circuit is then placed within the non-invasive control and mechanical action elements and is not handled thereafter. This significantly reduces the risk of compromising sterility over time. Regardless of the technical limitations overcome, the flowmeter according to the invention guarantees high accuracy over time for the experimenter.

[0071] The non-invasive flow meter may have applications outside the specific context described here, for example, outside the field of feeding bioreactors in culture media. Consequently, the liquid flow line could more generally take the form of any suitable conduit. When the bubble detectors are optical detectors, at least a portion of the conduit adjacent to the detectors transmits light.

[0072] While the systems in Figures 3 and 4 illustrate a single bioreactor 110; 110', it is intended that the culture medium can feed multiple bioreactors in parallel or a single bioreactor at a time, for example via a manifold and appropriate valve control.

[0073] Figure 6 illustrates such a system, comprising three bioreactors 210a-c in parallel, each supplied by a pump 230. Furthermore, flow initialization is achieved by means of valves 245a-c, forming a set of valves 244, operationally connected to a controller 250. The flow of a liquid matrix in the liquid flow line 240 to the bioreactors 210 is achieved by the pump 230. The initialization of the flow of the liquid matrix in the liquid flow line 240 to the bioreactors 210 is triggered – in an initialization phase – by the set of valves 244 between the pump 230 and the respective bioreactors 210a-c.As explained with reference to Figures 3 and 4, the syringe pumps 280a, b are actuated, preferably under the control of the controller 250, to inject the respective nutrients at precise flow rates, thus forming a culture medium having a mixture of a desired nutrient concentration. Preferably, the nutrients are injected at a rate that is a function of the measured liquid flow rate. The reservoir 260 can be filled via a pipe connected to the aqueous liquid source and through a filter 292, under the actuation of an inlet valve 241 and in response to signals from liquid level sensors 212, 214 on the reservoir 260, and under the control of the controller 250. The syringe pumps 280a, b can be filled from the respective reservoirs 282a, b with nutrients. While the system is illustrated here with two syringe pumps to add two respective nutrients (or nutrient mixtures), a system with multiple syringe pumps for additional nutrients can of course be envisioned.

[0074] The mixed culture medium can then be directed to provide a desired amount of culture medium to each 210a-c bioreactor, either simultaneously or sequentially. These dynamic capabilities allow access to fluctuating conditions that much more closely represent the natural environment and address metabolic guidance requirements in culture processes.

[0075] The sterility of the system is ensured by using a single liquid circuit that includes the flow line 240 from the reservoir 260 to the bioreactors 210a-c. Preferably, the inlet of the reservoir 260 is equipped with a biological filter 292, and the connections of the syringe pumps 280a, b to the flow line 240 are all sterile, removable connections, so that disconnecting a syringe pump from the line 240 does not affect the sterility of the circuit. It may also be possible to automatically refill the syringe(s) in a sterile manner, for example, if they are connected to a sterile nutrient reservoir. For example, solenoid valves allow switching from the nutrient suction circuit to the discharge circuit back to the flow line 240. LIST OF DIGITAL REFERENCES 10 bioreactors 12 low level sensor 14 high level sensor 20 tank 30 pump flow line culture medium controller aqueous liquid matrix reservoir nutrient concentrate reservoir flow meter nutrient feeding system bioreactor low level sensor high level sensor peristaltic pump flow line inlet valve culture medium valve set ac valves controller aqueous liquid matrix reservoir flow meter bubble injector; bubble front first detector a light source b light detector second detector a light source b light detector syringe pump a, b syringe pumps a, b nutrient concentrate reservoir filter 100' alternative nutrient feeding system for feeding into the culture medium of a bioreactor 110' bioreactor 130' peristaltic pump 140' line of debit 142' cultural environment 150' controller 160' aqueous liquid matrix reserve 170' flow meter 180' syringe pusher 190 filter 200 nutrient feeding systems for feeding culture media into a plurality of bioreactors 210a-c bioreactors 212 low level sensor 214 high level sensor 230 peristaltic pump 240 line of debit 241 inlet valve 244 valve sets 245a-c valves 250 controller 260 aqueous liquid matrix reserve 270 flow meter 280a, b syringe pumps 282a, b reserve of concentrated nutrient solutions 292 filter

Claims

CLAIMS 1. A method of supplying culture medium to a bioreactor (110), comprising: providing a source of liquid matrix (160); providing a liquid flow line (140) between the source of liquid matrix and the bioreactor; initiating a flow rate of the liquid matrix through the liquid flow line; measuring the flow rate of the liquid matrix through the liquid flow line using a non-invasive flow meter comprising a conduit of constant cross-section, a bubble injector adapted to inject one or more sterile bubbles into the conduit, a first optical sensor at a first position on the conduit downstream of the bubble injector and arranged to detect the passage of said one or more bubbles, and a second optical sensor at a second position on the conduit downstream of the first optical sensor and arranged to detect the passage of said one or more bubbles;wherein the flow rate is calculated based on a time difference between the second optical sensor and the first optical sensor respectively detecting the passage of said one or more bubbles; providing at least one source of enrichment substances (180); injecting at least one enrichment substance into the flow of the liquid matrix to form a culture medium (142) of defined concentration of enrichment substances in the liquid flow line; and providing a desired quantity of the culture medium to the bioreactor.; 2. Method according to claim 1, characterized in that the conduit is a portion of the liquid flow line (140).

3. Method according to any one of claims 1 and 2, characterized in that said at least one source of enrichment substances is in the form of at least one syringe pump (180) in sterile communication with the liquid flow line.

4. Method according to any one of the preceding claims, characterized in that said at least one enrichment substance is injected at a flow rate which is a function of the liquid flow rate measured by the flow meter.

5. A method according to claim any one of the preceding claims, characterized in that it is a method for supplying a culture medium to a plurality of bioreactors (210a-c) and in that the supply of a desired quantity of the culture medium comprises a preliminary step of initiating at least one flow rate of the liquid matrix by actuating a set of valves (245a to 245c) between the liquid flow line and the plurality of bioreactors.

6. Method according to the preceding claim, in which a culture medium having a dynamically adjustable concentration of enrichment substances is supplied to the bioreactor.

7. A system (100) for supplying enrichment substances for supplying culture medium to a bioreactor (110), comprising: a source of liquid matrix (160); a liquid flow line (140) between the source of liquid matrix and the bioreactor and means (130) for causing the liquid matrix to flow through the liquid flow line; at least one source of enrichment substances (180) in sterile connection with the liquid flow line; means for injecting said at least one source of enrichment substances (180) into the flow line to form a culture medium of defined concentration of enrichment substances in said flow line; and means for supplying a desired quantity of the culture medium to the bioreactor;a non-invasive flow meter comprising a conduit of constant cross-section, a bubble injector adapted to inject one or more sterile bubbles into the conduit, a first optical sensor at a first position on the conduit downstream of the bubble injector and arranged to detect passage; of said one or more bubbles, and a second optical sensor at a second position on the conduit downstream of the first optical sensor and arranged to detect the passage of said one or more bubbles; wherein the flow rate is calculated as a function of a time difference between the second optical sensor and the first optical sensor respectively detecting the passage of said one or more bubbles, the flow meter being configured to measure a flow rate of the liquid matrix through the liquid flow line.

8. The supply system according to the preceding claim, characterized in that the liquid flow line is part of a liquid circuit which is in one piece and which can be completely dismantled from the rest of the system.

9. The feed system according to any one of claims 7 and 8, characterized in that the means for causing the liquid matrix to flow through the liquid flow line comprises a pump (170), which is preferably a peristaltic pump.

10. The supply system according to any one of claims 7 to 9, characterized in that said at least one source of enrichment substances comprises at least one syringe pump (180) containing a respective enrichment substance and in sterile communication with the liquid flow line, preferably removably connectable to the liquid flow line via a sterile interconnection.

11. The supply system according to any one of claims 7 to 10, characterized in that the system further comprises a controller (150) operatively connected to each of the means (130) for causing the liquid matrix to flow through the liquid flow line, the means (180) for injecting the at least one source of enrichment substances, and the means for supplying a desired amount of the culture medium to the bioreactor, such that a culture medium (142) having a dynamically adjustable concentration of enrichment substances is supplied to the bioreactor in an automated manner.

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