Cell culture system with compartment and acoustic actuator and method thereof

Abstract

The present invention relates to a cell culture system comprising a flow compartment and a culture compartment separated by a membrane, the membrane being gas permeable and acoustically transparent, a culture chamber with at least one inlet and one outlet to the flow compartment and at least one inlet and one outlet to the culture compartment, and an acoustic actuator with a first element being an ultrasonic transducer and an opposing second element being a reflector or a second ultrasonic transducer, the acoustic actuator being arranged to generate an acoustic field in the culture compartment. The present invention also relates to a method for culturing cells using the cell culture system of the present invention.

Description

[Technical field]

[0001] The present invention relates to a cell culture system and a method thereof. In particular, the present invention relates to a cell culture system comprising a culture chamber with a flow compartment and a culture compartment, the cell culture system further comprising an acoustic actuator. The present invention relates to a method for culturing cells using such a cell culture system by maintaining at least one cell introduced into the culture compartment with the acoustic actuator. [Background technology]

[0002] Increasingly complex biological drugs such as antibodies, stem cells, and viral vectors are utilized in modern pharmacopoeias. These biological drugs are not produced by chemical synthesis, i.e. through the reaction of several chemical molecules, but by living cells. To reach the required quantities, these living cells must therefore be cultured in large quantities. These living cells are usually mammalian cell lines, and the traditional means of culture for mammalian cell lines is the bioreactor. Alternatively, insect cells have also been used in conjunction with the baculovirus expression vector system. Bioreactors are devices that automatically allow the cultivation of living organisms in growing quantities. These devices usually comprise a culture medium tank where stirring, incubation, oxygenation, and pH adjustment take place. The living organisms are usually contained in a culture medium in which the entire growth cycle of the organism takes place.

[0003] However, these bioreactors are designed for single-celled organisms such as yeast and bacteria, and are poorly adapted for cell culture of multicellular organisms. The problem is that the cells of multicellular organisms are fragile and are not used to growing alone by themselves. Conventional mammalian cells are usually grown surrounded by their peers in well-adjusted organisms such as humans.

[0004] In addition, many of the bioproduction capacities adapted to these multicellular organisms have been developed from bioreactors of bacteria and yeast, cells that are well suited to suspension culture in hostile environments. In contrast, cells from multicellular organisms, especially mammalian cells, are adherent cells, meaning that they usually grow attached to a substrate. Thus, a technological gap exists in this field between the demands and realities of biology.

[0005] Adherent culture techniques have been developed by parallel replication of usually simple validated culture strategies. For example, some techniques use adherent supports, and multi-layer / vertical stacking of culture surfaces. However, these techniques do not allow for sufficient production to meet the demand for biologics.

[0006] Antibodies were the first biologics produced on a large scale in history, and much of the literature on mammalian cell bioreactors is devoted to antibodies. However, traditional bioreactors are not suitable for all cell lines capable of producing the biologics of interest, and some adaptations must be made, as these bioreactors are not compatible with, for example, suspension cultures.

[0007] The first solution is to design cell lines adapted to culture in suspension in order to increase efficiency. Starting from an adherent cell line, it is generally possible to generate mutant clones adapted to suspension growth by adjusting the culture and feeding conditions (Berg DT et al., High-level expression of secreted proteins from cells adapted to serum-free suspension culture. Biotechniques. 1993 June; Mcallister, R. et al., Adaptation of Recombinant HEK-293 Cells to Growth in Serum Free Suspension, Animal Cell Technology: Products from Cells, Cells as Products: Proceedings of the 16th ESACT Meeting April 25-29, 1999, Lugano, Switzerland). Moreover, conventional suspension bioreactors (systems derived from bacterial cultures, such as for example US Pat. No. 10,640,741) can be used. This allows the volumetric growth of a large number of cells that are not limited by surface growth. At the end of the culture, it is necessary to remove the desired biologic from the culture mixture.

[0008] However, the process to obtain suspension-compatible cell lines is lengthy and costly, especially since the cell lines must originate from a single original clone that must be selected, tested, and stabilized from the original adherent cell line (Adaptation of Recombinant HEK-293 Cells to Growth in Serum Free Suspension. Mcallister R. et al., 1999, Animal Cell Technology: Products from Cells, Cells as Products. Springer, Dordrecht; Evolution from adherent to suspension: systems biology of HEK293 cell line development, Malm, M. et al., Sci Rep 10, 18996, 2020). This process can take multiple years to complete. Once a stable cell line has been engineered, there is still a problem with suspension culture of adherent cells, namely that these adherent cells tend to aggregate with each other very easily, which should be avoided (Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells, by Blessing D et al., Mol Ther Methods Clin Dev. 2018 Nov 22).

[0009] Another solution is to use carriers that allow volumetric culture in order to reduce the surface footprint of the culture of very large amounts of adherent cells. Providing a growth support in a volumetric approach can be done by adhering cells to microcarriers. For example, US20140356949 discloses a carrier for the expansion of induced pluripotent stem cells, comprising a substrate having one or more outer surfaces modified by gas plasma treatment and one or more structured indentations on the outer surface. Such carriers are then used in conventional suspension bioreactors.

[0010] Another approach could be to attach cells to 2.5-dimensional growth supports such as fibrous matrices or polymer foams, which are fractal scaffolds that provide many surfaces in 3-dimensional space (Valkama, A. et al., Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor, Nature, Gene Therapy 25, pp. 39-46, 2018).

[0011] However, when using microcarriers or in the case of solid substrates, volume loss is induced by the presence of the substrate. For this reason, carrier-based cultures produce lower titers than their suspension counterparts for an equal volume, which reduces the improvement. Furthermore, the use of supports for adherent cell lines often induces non-uniformity in the distribution of gases and nutrients in the case of perfusion bioreactors, as well as reduced culture density due to the space occupied by the growth support.

[0012] The existing solutions mentioned above are directed to the use of already developed bioreactors inherited from the cultivation of unicellular microorganisms. However, mammalian cells are fundamentally different and require a highly controlled environment. In particular, the shear rates present in the use of conventional stirred tank bioreactors, which are important for good distribution and mixing of gases and nutrients, are very lethal for these cell lines.

[0013] In the past, reactors using acoustic devices have been developed to hold molecules or cells in the reactor. For example, US Patent Application Publication No. 2016 / 0369236 specifically discloses a process for continuously harvesting cells from a cell culture, which is carried out in a bioreactor that includes a device for generating multi-dimensional standing waves that are used to hold the cell culture in place. A nutrient solution flow passes through the cell culture and circulates through the bioreactor to collect biological products generated from the cell culture. US Patent Application Publication No. 20180298323 and International Application Publication No. 2019140019 disclose an acoustic device that helps to hold molecules or cells, comprising an acoustic chamber or reactor and an ultrasonic transducer for creating multi-dimensional acoustic standing waves in the acoustic chamber. US Patent Application Publication No. 20170175073 discloses a system including a bioreactor and a primary clarification stage downstream of the bioreactor and having an acoustophoretic separator fluidly connected to the bioreactor. In such a system, the acoustophoretic separator comprises a flow chamber, an ultrasonic transducer, and an opposing reflector, which allow for the generation of multi-dimensional standing waves within the flow chamber.

[0014] In the state of the art, acoustic actuation is generally directly connected to the volume in which the biologic of interest is cultured. To allow the cells to be cultured at high density for long periods of time, the culture medium and gas must then be replaced. However, this imposes conditions on the strength of the acoustic field, since all liquid circulation must be disturbed by the acoustic field to retain its benefits. Furthermore, acoustic actuation of a liquid layer generates a large-scale recirculation in this liquid, commonly referred to as acoustic streaming (Jacob S. Bach and Henrik Bruus, Bulk-driven acoustic streaming at resonance in closed microcavities, Phys.Rev.E 100, 023104, 7 August 2019). This fluid motion can cause additional shear in fragile cells, or worse, can cause the cells to fall from their acoustic position if the acoustic field is not strong enough.

[0015] Therefore, there is a need for a system and method that facilitates the exchange of culture medium and gas without creating additional shear or any other mechanism that would affect the cultured cells.The present invention seeks to overcome the aforementioned shortcomings of the prior art, as it aims to prevent any disruption in the environment in which the cells are cultured, while providing a scalable technique for the cultivation of cells and the production of biopharmaceuticals. Summary of the Invention

[0016] To this effect, the present invention discloses a cell culture system comprising a culture chamber, the culture chamber including a flow compartment and a culture compartment separated by a membrane, the membrane being gas permeable and acoustically transparent, at least one inlet and one outlet to the flow compartment and at least one inlet and one outlet to the culture compartment, and an acoustic actuator comprising at least a first element being an ultrasonic transducer and an opposing second element being a reflector or a second ultrasonic transducer, wherein the acoustic actuator is positioned to generate an acoustic field in the culture compartment to maintain cells in the culture compartment.

[0017] Advantageously, the culture compartment of the cell culture system comprises a first end, a second end opposite the first end, and a main portion between the first end and the second end, the main portion of the culture compartment being at least partially located within the interior volume of the flow compartment.

[0018] Advantageously, the culture compartment of the cell culture system is located entirely within the interior volume of the flow compartment.

[0019] Conveniently, the distance between the acoustic actuator and the internal volume of the flow compartment is in the range 500 μm to 5 mm.

[0020] Conveniently, the distance between the first element and the second element of the acoustic actuator is determined according to equation (1):

[0021]

number

[0022] Advantageously, the membrane of the cell culture system is permeable to nutrients.

[0023] Advantageously, a wall of the culture chamber of the cell culture system is at least partially constituted by the acoustic actuator.

[0024] Advantageously, the membrane of the cell culture system comprises polyethylene glycol and / or polydimethylsiloxane.

[0025] Conveniently, the cell culture system is provided with a sampling inlet.

[0026] Advantageously, the acoustic actuator of the cell culture system further comprises a first element comprising a plurality of ultrasonic transducers and an opposing second element comprising a plurality of ultrasonic transducers and / or reflectors, wherein the plurality of ultrasonic transducers of the first element and the plurality of ultrasonic transducers and / or reflectors of the second element are arranged to generate an acoustic field and to maintain the cultured cells in the culture compartments at at least two positions spaced apart by a distance in the range of 50 μm to 500 μm. The separated cultured cells in the culture compartments may correspond to separated aggregates of cultured cells.

[0027] The present invention also discloses a set of cell culture systems as defined in any of the above, wherein the cell culture systems are parallelized.

[0028] The present invention also discloses a method for culturing cells using the cell culture system described above, comprising the steps of flowing a first liquid from an inlet of a flow compartment of a culture chamber to an outlet of the flow compartment and flowing a second liquid from an inlet of the culture compartment of the culture chamber to an outlet of the culture compartment, generating an acoustic field in the culture compartment using an acoustic actuator, introducing at least one cell in the culture compartment of the culture chamber, and maintaining the at least one cell introduced in the culture compartment.

[0029] Advantageously, the excitation frequency of the ultrasonic transducer of the acoustic actuator in the method for culturing cells is in the range of 500kHz to 15MHz and the excitation amplitude is in the range of 1W to 30W.

[0030] Conveniently, the method for culturing cells further comprises the step of temporarily setting an acoustic actuator to induce cavitation at the location where at least one cell is maintained.

[0031] Conveniently, the step of temporarily configuring the acoustic actuator to induce cavitation comprises reducing the frequency of the acoustic actuator to the range of 2 MHz to 3 MHz and increasing the amplitude to the range of 200 kHz to 400 kHz. [Brief description of the drawings]

[0032] The invention will be better understood, and its various features and advantages will become apparent, from the following description of several illustrative embodiments and the accompanying drawings, in which: [Figure 1] FIG. 1 shows a cross-section of a cell culture system comprising an acoustic actuator, a cell chamber comprising a flow compartment and a culture compartment. [Diagram 2] FIG. 2 shows a cross-section of a cell culture system comprising an acoustic actuator and a cell chamber comprising a flow compartment and a culture compartment, the main part of the culture compartment being located within the interior volume of the flow compartment. [Diagram 3] FIG. 3 shows a cell culture system comprising an acoustic actuator and a cell chamber comprising a flow compartment and a culture compartment, the culture compartment being located entirely within the interior volume of the flow compartment. [Figure 4] FIG. 4 shows a cell culture system comprising an acoustic actuator and a cell chamber comprising a flow compartment and multiple culture compartments. [Diagram 5] FIG. 5 shows a cross-section of a cell culture system comprising an acoustic actuator and a cell chamber comprising a flow compartment and a culture compartment. [Figure 6] FIG. 6 shows a cross section along axis A of the cell culture system described in FIG. [Figure 7] FIG. 7 shows a cross section along axis A of the cell culture system described in FIG.

[0033] Detailed Description of the Invention The present invention is described herein through examples, but is not limited to these examples.

[0034] FIG. 1 shows a cell culture system (10) comprising a culture chamber comprising a flow compartment (11) and a culture compartment (12), the culture compartment (12) having a first end (17), an opposing second end (18), and a main section (19) between the first end (17) and the second end (18). A gas-permeable membrane (13) separates the flow compartment (11) from the culture compartment (12). The culture chamber according to FIG. 1 also comprises an inlet (14a) and an outlet (14b) to the flow compartment (11) and an inlet (15a) and an outlet (15b) to the culture compartment (12). The cell culture system (10) also comprises an acoustic actuator comprising a first element (16a) which is an ultrasonic transducer and an opposing second element (16b) which is a reflector. The first and second elements of the acoustic actuator can be arranged to generate an acoustic field in the culture compartment. Such an acoustic field allows the cells (20) to be maintained in the culture compartment (12), where they progressively adhere to each other and thus provide each other with the necessary growth support. In Fig. 1 the first and second elements of the acoustic actuator are fixed on the flow compartment, but they may be located elsewhere, e.g. on the first and second ends of the culture compartment. Preferably, the elements of the acoustic actuator are positioned to generate an acoustic field in the center of the culture compartment, e.g. at the center of gravity, such that the cells (20) do not adhere to the membrane (13) and are thus maintained in suspension.

[0035] The cells in the culture compartment are not tied to a mechanical point and can be manipulated in the volume of the culture compartment by adjusting the phase of the acoustic waves created by the acoustic actuator. For example, adjusting the phase of the acoustic waves allows the cells to move to a sample outlet, which can correspond to the inlet (15a) or outlet (15b) of the culture compartment or another outlet (not shown) that is connected to the volume of the culture compartment.

[0036] With the flow and culture compartments of the invention, the volume is physically delimited by a wall or by a membrane at the separation between the flow and culture compartments. Preferably, the separation between the flow and culture compartments is constituted by a membrane (13), but such separation may partially comprise a wall. Preferably, but not limited to the cell culture system described in FIG. 1, these walls are biocompatible, do not release molecules into the liquid they are in contact with, are acoustically transparent, and can be manufactured by injection or molding. For example, such walls may be made of materials including polypropylene or polystyrene, or obtained from polymer resins adapted for stereolithography.

[0037] The inlet (14a) allows for the introduction of a liquid into the flow compartment (11), which is then discharged through the outlet (14b). The liquid introduced into the flow compartment may include gas or nutrients and combinations thereof. The inlet (15a) allows for the introduction of a liquid into the culture compartment (12), which is then discharged through the outlet (15b). The liquid introduced into the culture compartment may include gas or nutrients and combinations thereof. In particular, the inlet (15a) of the culture compartment (12) allows for the introduction of one or more cells into the culture compartment. The outlet (15b) of the culture compartment (12) allows for the collection of one or more cells that may have been previously introduced and maintained and cultured in the culture compartment. By "cultured cells" is meant cells that have been maintained in the culture compartment (12) and may, for example, undergo proliferation, differentiation, or maturation.

[0038] In FIG. 1, the culture compartment (12) has a first end (17), a second end (18) opposite the first end, and a main part (19) between the first and second ends. The main part (19) extends from the first end to the second end, as indicated by the double arrow in FIG. 1, and corresponds to the culture compartment, except for the first end (17) and the second end (18) and the inlet and outlet (15a) and (15b). The main part (19) of the culture compartment (12) is partially located within the internal volume (11b) of the flow compartment (11), while the first end (17) and the second end (18) of the culture compartment are not located within the internal volume of the flow compartment. Preferably, the exchange surface between the culture compartment and the flow compartment corresponds to the membrane separating these compartments and is as large as possible in order to maximize the exchange between the liquids in the flow compartment and the culture compartment. Although not essential, when at least the main part of the culture compartment is partially located inside the inner volume (11b) of the flow compartment (11), the exchange surface between the culture compartment and the flow compartment is increased.

[0039] In FIG. 1, a first sensor group (21) is located in the flow compartment (11) and a second sensor group (22) is located in the culture compartment (12). These sensor groups can be located at different positions in the corresponding compartments. Alternatively, the first and / or second sensor groups may be located at the inlet and / or outlet of the flow or culture compartment. Furthermore, the sensor groups can be connected to one or more controllers located outside the compartments. The connection between the sensor groups and the controllers may include wires or may be wireless. The sensor groups (21) and (22) can monitor pH, temperature, dissolved oxygen, carbon dioxide, pressure, flux rate, shear rate, etc.

[0040] The cell culture system (10) of FIG. 1 allows the introduction of a liquid containing at least one cell (20) into the culture compartment (12) through the inlet (15a) and the maintenance of the at least one cell in the culture compartment by the first and second elements (16a) and (16b) of the acoustic actuator. The at least one cell (20) is provided with a gas for culture, differentiation or maturation by a liquid supplied through the inlet (14a) of the flow compartment, the gas passing through the membrane (13). The membrane (13) prevents the turbulent flow (11a) generated in the flow compartment (11) by the supply of the liquid from the inlet (14a) from being transmitted into the culture compartment (12). In fact, the turbulent flow (11a) generates shear in the environment of the cultured cells, causing mechanical or biological stresses to the cells, affecting their proliferation, differentiation or maturation. In the worst case, depending on the cells being cultured, the shear kills some of the cells. In particular, the cell culture system of the present invention does not require impellers or blades to mix the contents of the culture compartments.

[0041] Thus, liquid introduced through the inlet (14a) at a feed rate that creates turbulence can be continuously introduced into the flow compartment, with the membrane preventing the turbulence from being sent to the culture compartment. Furthermore, because the feed rate is not limited by the turbulence that can be created, replacement of the liquid in the flow compartment can be performed faster, and the gas supplied to the culture compartment can be more and more precisely and / or quickly adjusted. Furthermore, the liquid introduced into the flow compartment can contain bubbles without harming the cells. Bubbles can be introduced into the flow compartment, for example, into the liquid introduced through the inlet of the flow compartment. Bubbles can increase the gas exchange between the liquid introduced into the flow compartment and the liquid already present in the flow compartment. Thus, the increase in gas exchange in the flow compartment can also increase the gas exchange between the flow compartment and the culture compartment. Bubbles during acoustic operation are usually to be avoided, since they generate high shear forces when vibrating under acoustic actuation. However, according to the cell culture system of the present invention, the membrane prevents any shear forces from being sent to the culture compartment and the cells.

[0042] Furthermore, the liquid in the flow compartment can be replaced more quickly, allowing for more precise and / or faster adjustment of temperature, pH, oxygen and carbon dissolved, and chemical concentrations in the flow and culture compartments. The culture system of the present invention allows for replacing the gas content in the liquid in the culture compartment while avoiding changing the flux rate in the culture compartment.

[0043] Although not shown in Fig. 1, in the three-dimensional cell culture system described in Fig. 1, in particular the culture chamber may have a right circular cylinder, a square cylinder, or any other shape of a sphere. The culture chamber may also have a rectangular parallelepiped shape, such as a rectangular prism, thereby including flat parallel surfaces onto which the acoustic actuator may be fixed.

[0044] FIG. 2 shows a cell culture system (20) comprising a culture chamber with a flow compartment (11) and a culture compartment (12), the culture compartment having a first end (17) and an opposing second end (18) and a main part (19) between the first and second ends of the culture compartment. A gas-permeable membrane (13) separates the flow compartment (11) from the culture compartment (12). The culture chamber according to FIG. 2 also comprises an inlet (14a) and an outlet (14b) to the flow compartment and an inlet (15a) and an outlet (15b) to the culture compartment. The cell culture system also comprises a first element (16a) which is an ultrasonic transducer and an opposing second element (16b) which is a reflector. The first and second elements are arranged so that they can generate an acoustic field in the culture compartment. In FIG. 2, the first end, the second end and the main part of the culture compartment are placed inside the internal volume (11b) of the flow compartment (11). The cell culture system also includes similar groups of sensors (21) and (22), as in FIG. 1, which are located in the flow compartment (11) and the culture compartment (12).

[0045] FIG. 3 comprises an acoustic actuator comprising a first element (16a) and an opposing second element (not visible) and a culture chamber comprising a flow compartment (11) and a culture compartment (12), the culture compartment being located entirely within the internal volume (11b) of the flow compartment (11). FIG. 3 shows only a portion of the cell culture system to illustrate the culture compartment within the flow compartment. A membrane (13) separates the volume of the flow compartment from the volume of the culture compartment. In FIG. 3, the flow compartment has the shape of a right circular cylinder, which surrounds the culture compartment, which also has the shape of a right circular cylinder, the flow compartment and the culture compartment sharing the same axis. Thus, the flow compartment (11) can be provided with a liquid containing gas, whereby the gas is delivered through the membrane (13) to the liquid in the culture compartment to feed the cells.

[0046] FIG. 4 shows a cell culture system (40) comprising an acoustic actuator comprising a first element (16a) and an opposing second element (not visible) and a cell chamber comprising a flow compartment (11) and a culture compartment (12), the culture compartment comprising a number of right circular cylinders with an axis parallel to the axis of the flow compartment. All the right circular cylinders of the culture compartment are located entirely within the inner volume (11b) of the flow compartment (11). A membrane (13) separates the volume of the flow compartment from the volume of the number of right circular cylinders of the culture compartment. In FIG. 4, the flow compartment surrounds the number of right circular cylinders of the culture compartment. Such an embodiment allows for the simultaneous growth of different types of cells while supplying the gases required for the cell culture using only the liquid provided in the flow compartment. Each right circular cylinder of the culture compartment may be connected to an inlet and an inlet or may share the same inlet and outlet.

[0047] FIG. 5 shows a cell culture system (50) comprising a culture chamber with a flow compartment (11) and a culture compartment (12), the culture compartment having a first end (17) and an opposing second end (18) as well as a main part (19) between the first and second ends of the culture compartment, indicated by a double arrow. A gas-permeable membrane (13) separates the flow compartment (11) from the culture compartment (12). In other words, the interface between the flow compartment and the culture compartment is at least partially made of the membrane (13). The culture chamber according to FIG. 5 also comprises an inlet (14a) and an outlet (14b) to the flow compartment and an inlet (15a) and an outlet (15b) to the culture compartment. The cell culture system also comprises an acoustic actuator comprising a first element (16a) which is an ultrasonic transducer and an opposing second element (16b) which is a reflector. The first and second elements are arranged so that an acoustic field can be generated in the culture compartment. In Fig. 5, the first and second ends of the culture compartment do not constitute an interface between them and the flow compartment (11), but the main part of the culture compartment extending along the double arrow faces the flow compartment, i.e., in other words constitutes an interface between the flow compartment and the main part. The cell culture system also includes a group of sensors (21) and (22) similar to Fig. 1, which are arranged in the flow compartment (11) and the culture compartment (12), respectively. Unlike the cell culture systems described in Figs. 1 and 2, the main part (19) of the culture compartment is not located inside the inner volume (11b) of the flow compartment (11), but faces the flow compartment side-by-side. In other words, the flow compartment and the culture compartment are adjacent or adjacent to each other between at least a part of the main part of the culture compartment.

[0048] The elements of the cell culture system described in Figure 5 are arranged such that the actuator generates an acoustic field in the center of the culture compartment, which keeps the cells (20) in the culture compartment (12) without adhering to the membrane (13) and therefore in suspension.

[0049] In the cell culture system (50) shown in FIG. 5, the main part of the culture compartment, represented along the arrow (21), is not located inside the internal volume (11b) of the flow compartment (11), but faces the flow compartment. The interface between the main part of the culture compartment and the flow compartment is at least partially formed by the membrane (13). or comprises a membrane (13).

[0050] FIG. 6 shows a cross section along the A axis of the cell culture system described in FIG. 1. Although represented in FIG. 6 as having a spherical shape of a right cylinder, the cell culture system of the present invention is not limited to such a right cylinder shape, other shapes are possible, such as a square cylinder or any other shape. The cell culture system described in FIG. 6 comprises an acoustic actuator comprising a first element (16a) which is an ultrasonic transducer and an opposing second element (16b) which is a reflector or another ultrasonic transducer. The first and second elements are arranged to generate an acoustic field in the culture compartment (12). In FIG. 6, the culture compartment (12) is placed inside the inner volume (11b) of the flow compartment (11). The membrane (13) prevents turbulence (11a) generated in the flow compartment (11) by the supply of liquid being pumped into the culture compartment (12) where the cells (20) are maintained by the acoustic actuator.

[0051] FIG. 7 shows a cross section along the B axis of the cell culture system described in FIG. 5. Although represented in FIG. 7 as having a spherical shape of a right cylinder, the cell culture system of the invention is not limited to such a right cylinder shape, other shapes are possible, such as a square cylinder or any other shape. The cell culture system described in FIG. 7 comprises an acoustic actuator comprising a first element (16a) which is an ultrasonic transducer and an opposing second element (16b) which is a reflector or another ultrasonic transducer. The first and second elements are arranged to generate an acoustic field in the culture compartment (12). In FIG. 7, the culture compartment is adjacent to the flow compartment, the interface between them at least partially consisting of or including the membrane (13). The membrane (13) prevents turbulence (11a) generated in the flow compartment (11) by the supply of liquid being pumped into the culture compartment (12) in which the cells (20) are maintained by the acoustic actuator.

[0052] The membrane (13) of Figures 1 to 7 is acoustically transparent, i.e. its acoustic impedance is the same as the liquid in the culture compartment. In certain embodiments, the membrane is also permeable to nutrients, which allows the introduction of nutrients in the flow compartment to diffuse through the membrane into the culture compartment. The membrane may be flexible, so that the culture compartment can be a bag floating in the internal volume of the flow compartment. Preferably, the membrane (13) is made of a biocompatible material. The membrane (13) may comprise polyethylene glycol and / or polydimethylsiloxane (PDMS) or polyethylene glycol and / or polydimethylsiloxane (PDMS).

[0053] In Figures 1, 2 and 5, the main part (19) extends from a first end (17) to an opposite second end (18) and is represented by an arrow. In other words, the first end (17) and the opposite second end (18) consist of or include at least a wall of the membrane (13) or a part of the membrane (13) that separates the culture compartment from the outer wall or flow compartment of the cell culture system, or in some embodiments even from the actuators. The main part (19) extends from the first end to the opposite second end and represents both the wall at least partially consisting of the membrane and the inner volume of the culture compartment. The inlet part (15a) and the outlet part (15b) of the culture compartment are not part of the main part. In other words, the main part corresponds to the culture compartment, except for the ends flanking this culture compartment as well as the inlet and outlet of the culture compartment.

[0054] Preferably, the distance between the first and second elements of the acoustic actuator is dependent on the acoustic frequency used and is determined by equation (1):

[0055]

number

[0056] Preferably, the thickness of the wall of the flow section adjacent to the ultrasonic transducer of the acoustic actuator depends on the acoustic frequency used and is determined by Equation (2):

[0057]

number

[0058] Preferably, the cell culture system of the invention comprises a total volume of the flow and culture compartments of 1 milliliter to 1000 liters, more preferably 200 liters to 1000 liters. Cell culture systems with a total volume of 1 mL to 250 mL are particularly adapted for parametric exploration or paralleling experiments to optimize processes. Culture systems with a total volume of 1 L to 80 L are particularly adapted for preclinical production, animal testing or small-scale therapy. Culture systems with a total volume of 150 L or more are particularly adapted for industrial production and clinical trials gene therapy.

[0059] Preferably, the ultrasonic transducer of the present invention comprises a piezoelectric ceramic such as PZT ceramic, but the present invention is not limited to PZT ceramic. According to the system of the present invention, an excitation frequency in the range of 100 kHz to 20 MHz and preferably 500 kHz to 15 MHz is applied by the ultrasonic transducer. Furthermore, the excitation frequency is in the range of 1 W to 1000 W. More specifically, the excitation frequency is a minimum of 1.5 W per liter. Thus, for a 1 liter cell culture system, the excitation frequency is 1.5 W to 30 W, for a 10 liter cell culture system, the excitation frequency is 15 W to 200 W, and for a 100 liter cell culture system, the excitation frequency is 150 W to 1000 W. The ultrasonic transducer of the acoustic actuator can also be flexible so that it can conform to the shape of the flow compartment of the cell culture system.

[0060] For example, typical dimensions of an ultrasonic transducer of the first or second element of an acoustic actuator are 70 x 70 mm, with a thickness dependent on frequency, being 0.5 mm at a frequency of 4 MHz, 1 mm at a frequency of 2 MHz, and 2 mm at a frequency of 1 MHz.

[0061] The reflector of the second element of the acoustic actuator has a significant impedance break with the medium in the culture compartment. Thus, it is preferable to use a material such as stainless steel 316 or iron with a thickness of 8 g / cm as used in the biomedical industry. 3 density of 100, speed of sound of 5521 m / s, and 44.2 MPa s / m 3 or 2.7 g / cm 3 density of 100, sound speed of 6108 m / s, and 16.5 MPa s / m 3 A material with a high acoustic impedance, such as aluminum, which has an acoustic impedance of 100 .mu.m, is preferred.

[0062] Preferably, the membrane has a thickness of 50 μm to 2 μm. Preferably, the membrane has a pore size in the range of 0.2 μm to 4 μm, which prevents the cultured cells from escaping from the culture compartment.

[0063] Preferably, the sensor is configured to monitor multiple parameters and provide an alert when a parameter threshold is exceeded, for example pH should be in the range of 6-8, temperature 30°C-40°C, dissolved oxygen 0mg / L-20mg / L, carbon dioxide 0mg / L-100mg / L, pressure 0bar-1bar, and flux rate 0L / min-20L / min.

[0064] In certain embodiments, which may be combined with other embodiments disclosed herein, the first and / or second elements of the acoustic actuator may be integrated into a wall of the culture compartment, for example into a wall of the flow compartment or the culture compartment. Alternatively, the acoustic actuator may form part of the wall of the flow compartment of the culture compartment.

[0065] The cells cultured in the cell culture system of the invention may be bacteria, e.g. cyanobacteria, plant cells, e.g. algae, animal cells (human or non-human), e.g. mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, T lymphocytes (cart-T), insect cells or mammalian cells, e.g. human embryonic kidney cells (HEK), or others. In the case of human embryonic kidney cells, they are necessarily obtained without destroying the embryos from which they are derived and are not suitable for inducing human developmental processes.

[0066] The system of the present invention is associated with other similar systems of the present invention as a set of systems, the systems being paralleled to increase the overall capacity of the overall set of systems. In a preferred embodiment, these systems have a cuboid shape to allow them to be stacked, in particular to minimize the space they occupy. In the set of systems, groups of sensors can be connected to one or more controllers to manage the overall set of systems or specific systems of the systems of the set of systems. EXAMPLES

[0067] In a particular embodiment, the cell culture system comprises an acoustic actuator comprising a first element comprising three ultrasonic transducers and an opposing second element comprising three reflectors, each reflector facing an ultrasonic transducer so as to generate an acoustic field in the culture compartment. The ultrasonic transducers and reflectors are 70×70 mm in size and are arranged alternately over a length of 210 mm. The thickness of the walls of the flow compartment is calculated according to equation (3):

[0068]

number

[0069] In this example, the membrane prevents the propagation of liquid turbulence from the flow compartment to the culture compartment and is made of polydimethylsiloxane (PDMS), although other materials may be used for the membrane.

Claims

1. A cell culture system (10), comprising: A culture chamber, a flow compartment (11) and a culture compartment (12) separated by a membrane (13), said membrane (13) being gas permeable and acoustically transparent; at least one inlet (14a) and one outlet (14b) to said flow section (11); at least one inlet (15a) and one outlet (15b) to said culture compartment (12); a culture chamber, An acoustically actuated device comprising at least a first element (16a) that is an ultrasonic transducer and an opposing second element (16b) that is a reflector or a second ultrasonic transducer. Equipped with wherein the acoustic actuator is arranged to generate an acoustic field in the culture compartment (12) to maintain cells in the culture compartment (12). A cell culture system (10).

2. the culture compartment (12) comprises a first end (17), a second end (18) opposite the first end (17), and a main portion (19) between the first end (17) and the second end (18); 2. The cell culture system (10) according to claim 1, wherein the main part (19) of the culture compartment (12) is at least partially located inside the inner volume (11b) of the flow compartment (11).

3. 3. The cell culture system (10) of claim 2, wherein the culture compartment (12) is located entirely within the inner volume (11b) of the flow compartment (11).

4. The cell culture system (10) of claim 1, wherein the distance between the acoustic actuator and the inner volume (11b) of the flow compartment (11) is in the range of 500 μm to 5 mm.

5. The distance between the first element (16a) and the second element (16b) of the acoustically actuated device is calculated by equation (1): [Equation 1] in accordance with In the formula, h ｔｏｔ 2. The cell culture system (10) of claim 1, wherein λ is the distance between the first and second elements (16a) and (16b) of the acoustic actuator, f is the acoustic frequency used, c is the propagation speed of sound waves in liquid between the first and second elements (16a) and (16b) of the acoustic actuator, and λ is the wavelength of the sound waves.

6. The cell culture system (10) of claim 1, wherein the membrane (13) is permeable to nutrients.

7. The cell culture system (10) of claim 1, wherein a wall of the culture chamber is at least partially composed of the acoustic actuator.

8. The cell culture system (10) of claim 1, wherein the membrane (13) comprises polyethylene glycol and / or polydimethylsiloxane.

9. The cell culture system (10) of claim 1, comprising a sampling inlet.

10. the acoustically actuated device further comprises a first element comprising a plurality of ultrasonic transducers (16a, 16c, 16e, 16g) and an opposing second element comprising a plurality of ultrasonic transducers and / or reflectors (16b, 16d, 16f, 16h); 2. The cell culture system (10) of claim 1, wherein the plurality of ultrasonic transducers of the first element and the plurality of ultrasonic transducers and / or reflectors of the second element are arranged to generate an acoustic field and maintain cultured cells (20) in the culture compartments (12) at at least two positions spaced apart by a distance in the range of 50 μm to 500 μm.

11. 10. The set of cell culture systems as defined in claim 1, wherein said set of cell culture systems is parallelized.

12. A method for culturing cells using a cell culture system (10) according to any one of claims 1 to 10, comprising: Flowing a first liquid from the inlet (14a) of the flow compartment (11) of the culture chamber to the outlet (14b) of the flow compartment (11), and flowing a second liquid from the inlet (15a) of the culture compartment (12) of the culture chamber to the outlet (15b) of the culture compartment (12); generating an acoustic field in the culture compartment (12) using the acoustic actuator; Introducing at least one cell into the culture compartment (12) of the culture chamber; maintaining said at least one cell introduced into said culture compartment (12); A method comprising:

13. 13. The method of claim 12, wherein the excitation frequency of the ultrasonic transducer of the acoustically actuated device is in the range of 500 kHz to 15 MHz and the excitation amplitude is in the range of 1 W to 30 W.

14. 13. The method of claim 12, further comprising temporarily configuring the acoustic actuator to induce cavitation at a location where the at least one cell is maintained.

15. 15. The method of claim 14, wherein temporarily configuring the acoustic actuator to induce cavitation comprises decreasing the frequency of the acoustic actuator to a range of 2 MHz to 3 MHz and increasing the amplitude to a range of 200 kHz to 400 kHz.

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