Device and method using pulsed electromagnetic fields to alter the state of a product and / or the production of said product

Abstract

The present invention relates to an apparatus and method that allows for applying an electromagnetic field to a product over a period of time to change the state of the product. This change in state can accelerate an ongoing process and / or alter the quality of the experience of subsequent use of the product. The apparatus comprises a support and a container in which the product is located, wherein the support comprises one or more modules for generating a pulsed electromagnetic field (PEMF), and the support and thus the modules are connected to a control device for controlling the generation of the PEMF and are positionable relative to the product to expose the product to the generated PEMF.

Description

Technical Field

[0001] The invention to which this application relates is the application of pulsed electromagnetic fields (PEMFs), which may also be referred to as electromagnetic digital sequences, to provide a change in product state, and more specifically, to alter one of the metabolic productivity properties of a biological system (e.g., fermentation and cell culture biological systems) and / or to increase the productivity of such a system. Background Art

[0002] In the field of microbial cultivation, development has been underway for many years to produce a wide variety of foods and beverages for human or animal consumption. For example, the fermentation process of yeast (Saccharomyces species) is a key part of the production process for beer, wine, and leavened bread. The development of this form of food manufacturing was initially based on the serendipitous discovery of natural cultures, which were subsequently adopted in production processes. Since then, improvements in knowledge about the management of fermented product production have meant that development has begun, but it is still largely based on "trial and error" and involves observing the production processes used and then learning from their mistakes.

[0003] More recently, development processes have become more standardized and scientific, but arguably, much of the scientific progress has been related to avoiding spoilage and recovering from mistakes, rather than improving the productivity of the primary fermentation itself. Nevertheless, many production methods, such as winemaking, still rely on the natural yeasts present on the surface of grapes, and despite improvements in cleanliness and modern vessel design, the primary fermentation processes used remain very close to those used in ancient times. Similarly, the production of beer, cheese, and fermented bread has not changed substantially from the original primary microbial processes used.

[0004] Furthermore, when carbon dioxide is included in beverages, it adds an extra dimension to the liquid's taste, texture, and thirst-quenching properties. The gas can be added directly by spraying carbon dioxide into the liquid, or it can be provided by the action of yeast and dissolved sugars. In some cases, such as keg and bottled beer, both methods of carbon dioxide addition can be used. Many liquid beverages utilize the addition of carbon dioxide to provide effervescence, thereby enhancing and expanding the taste and texture of the drinking experience, such as non-alcoholic fruit and sugar-based liquids and alcohol-based beverages. In all of the above aspects, it is assumed that the carbon dioxide is thoroughly mixed with the aqueous medium, but it has been found that, particularly when alcohol is also present in the mixture, conventional mixing achieved at the molecular level has a negative impact on the overall drinking experience. This is believed to be due to water's natural tendency to form irregular intermolecular hydrogen bonds, which result in clusters randomly distributed in the medium. Similarly, alcohol tends to cluster, and this results in a less-than-ideal distribution of carbon dioxide in the beverage product. As a result, certain aspects of the liquid, such as the mousse in champagne, which is the mouthfeel of carbon dioxide mixed with water and alcohol, cannot be achieved to the desired degree. Another problem is that the addition of gas (especially by sparging) can lead to excessive disruption of the liquid's open hydrogen-bonded structure and thus lead to clumping. Conventionally, the solution is to store the liquid in a container for a long time to allow natural kinetic movement to homogenize the system. This can require years of expensive storage time for the product to regain the preferred open structure of the liquid, in which the gas and alcohol can be evenly contained.

[0005] In recent times, much of the skills and experience gained from brewing and winemaking have been applied to the production of, for example, biopharmaceuticals, where the fermentation systems and equipment employed are largely similar, but must comply with relatively strict regulatory parameters and the organisms used have been genetically modified. However, once again, the growth and performance of the culture still fundamentally depends on the inherent behavior of the original organism. It has been found that these processes can be optimized through judicious selection of nutrients and careful control of temperature, gas exchange, and / or other batch conditions, but it has been found that microbial productivity cannot exceed the natural limits of the microorganisms involved.

[0006] Furthermore, a common factor in all of the above processes (both modern and older) is that a period of time is required between the start of the process and the end of the process in order to allow the yeast and / or other organisms to fully develop their functions in the product. This time delay is a significant obstacle to producing the product in the desired form on a larger scale and more efficiently, and / or, if insufficient time is available to perform the full function, it may mean that the quality of the final product is poor. Consequently, commercially significant productivity is limited or unattainable, as it is believed that microbial cultures have now reached natural limits of productivity and that this can be achieved by optimizing nutrients, growth conditions, and / or equipment. Consequently, it is generally considered difficult to change the process used for a particular product without compromising quality and / or violating regulations.

[0007] For example, in the area of mammalian cell culture, which is used across many areas of the medical and biotechnology industries to produce a wide range of products, including enzymes, hormones, and antibodies, the production of biologics using mammalian cells is typically very expensive due to the slow cell growth rate, highly specialized state, and higher risk of contamination compared to traditional microbial systems, yet this conventional approach is considered the only viable solution.

[0008] The applicant disclosed in its co-pending application PCT / GB2018 / 053493 (the contents of which are incorporated herein) the ability to provide an electromagnetic field in pulsed form and expose it to certain products to alter the metabolic productivity of liquid biological systems, such as fermentation and cell culture.

[0009] However, for this application to be effective it is necessary to be able to ensure that the electromagnetic field is applied in a reliable and repeatable manner to ensure that the effect of the method is achieved each time the liquid is exposed to the pulsed electromagnetic field. Summary of the Invention

[0010] One object of the present invention is therefore to provide a solution to the above-mentioned problems which allows to improve the quality and process for obtaining the product in the developed and desired form, thereby increasing the quality of the final product and / or speeding up the way to achieve the final product. Another object is therefore to provide a method which is non-invasive, easy to apply and which can provide an increased yield and / or reduce the batch production time.

[0011] Another object of the present invention is to provide a device that allows an electromagnetic field to be effectively applied to a product in an easily reproducible manner and that can preferably be performed by non-technical personnel, if desired. Another object is to provide a device in a form that allows it to be used in combination with a container containing the product to be treated.

[0012] In a first aspect of the invention, there is provided a device allowing application of an electromagnetic field to a product for a period of time in order to change the state of said product, said device comprising at least one support and a container, said product being positioned in said container, and wherein said support comprises one or more modules for generating a pulsed electromagnetic field (PEMF), and said support comprises or is connected to control means for controlling the generation of the PEMF and is positionable relative to said product in order to expose the product to said generated pulsed electromagnetic field.

[0013] Typically, the device is provided to allow for the delivery of PEMF to facilitate thorough mixing of the components of the product.

[0014] In one embodiment, the device controls the frequency and digital sequence of the emitted PEMF to correspond to the dielectric and / or other characteristics of the product then held in the container.

[0015] In one embodiment, the control means is provided in the form of an integrated circuit provided on the support and may include a transmitter to allow emission of PEMF from the transmitter in addition to the PEMF emitted from the module.

[0016] In one embodiment, the control device is in turn operable via a software-based user interface to allow a user to control the generation of PEMF from the device.

[0017] In one embodiment, the support and / or module is positionable relative to the container to allow exposure of a product held in the container to the PEMF.

[0018] In one embodiment, a plurality of such modules are arranged on a support in a fixed array or configuration to provide PEMF of increasing range and / or intensity.

[0019] In one embodiment, the support is located outside of the container, and the PEMF is applied to the product through one or more walls of the container in which the product is located.

[0020] In an alternative embodiment, at least a portion of a support comprising one or more modules for generating PEMF is located inside the container.

[0021] Typically, multiple supports may be located at different locations within the container to provide uniform exposure to the PEMF generated by the modules with which the supports are positioned.

[0022] In one embodiment, the support is formed by one or more walls of the container and the module is mounted as part of the one or more walls. In an alternative embodiment, the support is located within one or more walls of the container.

[0023] In one embodiment, the support is provided in the form of a housing in which the one or more modules are located, or in another embodiment, the support is provided in the form of a sheet on which the modules are located.

[0024] In one embodiment, the support is provided in a sterile form for use, and in one embodiment may be provided for single use.

[0025] In one embodiment, the module includes an antenna and a transmitter to allow for wireless short-range communication of PEMF within a specific frequency range. In one embodiment, the specific frequency range is the Industrial, Scientific, and Medical (ISM) short-range radio band. In one embodiment, the frequency is 2.4 GHz.

[0026] In one embodiment, the transmitter is capable of generating PEMF at a distance of up to 15 meters.

[0027] In one embodiment, the control device allows PEMF to be delivered in pulses having a duration in the range of 0.5-1.5 ms, and / or the pulses are separated by rest periods in the range of 40-66 ms, and / or the PEMF pulses are emitted in the range of 12-20 pulses per second.

[0028] Typically, the support and / or the modules located thereon are arranged relative to the container so as to generate the PEMF in an omnidirectional manner relative to the product.

[0029] In one embodiment, the module is based on a personal area network system device.

[0030] In one embodiment, the control device and the PEMF emitting modules are provided with a support in the form of a radio-transparent housing in which the modules are located, and the shape of the housing and the spacing of the modules may be adapted to allow their use in association with one or a range of container types.

[0031] In one embodiment, the housing, and therefore the device, is provided as an integral part of another article that can be used with the container, or formed as part of a container that holds a liquid.

[0032] In one embodiment, the support is shaped, e.g. moulded to the contours of a particular container with which it is to be used, such that the positioning means of the device allow the container to fit securely therewith, thereby in one embodiment allowing the device to be used immediately before the product is consumed.

[0033] In one embodiment, the container is either a single bottle or cup, or may be a group of containers, such as multiple bottles or cups, and contains a product therein, and in one embodiment the product is a sparkling liquid and / or contains alcohol, such as champagne, Prosecco, Cava, etc.

[0034] In another embodiment, the container may be in the form of a bioreactor vessel, and it will be understood that the container used is arranged in a form suitable for the product to be contained therein and / or the processing steps to be performed on the product, and that the PEMF is selectively applied to the product as an additional step or in at least one step.

[0035] In one embodiment, the apparatus is provided with positioning means allowing the base of a container to be placed thereon and / or may be provided with engaging means to be placed around the container.

[0036] In one embodiment, the device is provided in the form of a housing that fits over the neck of a bottle.

[0037] Typically, the device includes a battery or other power supply means and / or can be recharged to allow power to be provided to emit the electromagnetic field pulses.

[0038] In another embodiment, the device is provided in the form of a sleeve which can be placed around the container and which can also be provided with means to allow cooling of the liquid in the container.

[0039] In one embodiment, the device includes at least one feature that allows for changing the visual appearance of the container, such as providing lighting for the device to provide additional visual dimension when the container is a cup, and / or indicating the operation of the device and when and when PEMF is being generated.

[0040] In another aspect of the present invention, a method for changing the state of a product is provided, wherein the method comprises the following steps: applying a pulsed electromagnetic field from one or more modules to a product when in a first state at a predetermined frequency and for a predetermined time period to change the product in the first state into another desired product for subsequent use or further processing.

[0041] In one embodiment, the change in state is a result of fermentation and / or development of the cell culture system in which the product is produced.

[0042] In one embodiment, the PEMF allows for a change in state of one or more components of a product, which are in the form of elements or ingredients of the product.

[0043] In one embodiment, PEMF is used as a stage in product processing to induce fermentation and / or cell culture development in the product.

[0044] In one embodiment, the change of state is to increase the speed at which a processing step of the product occurs. In one embodiment, the processing step is the development of a cell culture.

[0045] In one embodiment, PEMF is applied to increase the growth rate of a culture of mammalian cells.

[0046] In one embodiment, PEMF is intentionally not applied to the product during other stages of its processing.

[0047] In one embodiment, the PEMF is applied for a predetermined period of time, which is determined with reference to a particular product and / or amount of product.

[0048] In one embodiment, the PEMF frequency is within the bandwidth of the electromagnetic spectrum used for industrial scientific and medical purposes.

[0049] In one embodiment, the electromagnetic energy is delivered in pulses having a duration in the range of 0.5-1.5 ms.

[0050] In one embodiment, the pulses are separated by rest periods in the range of 40-66 ms.

[0051] In one embodiment, a plurality of the devices are arranged in a fixed array or selectively positioned in an array to provide a stronger pulsed magnetic field or a pulsed magnetic field with a greater range.

[0052] In one embodiment, PEMF is applied simultaneously to products held in multiple containers using an apparatus having the containers positioned in a specific array.

[0053] In one embodiment, the container is a bottle containing a sparkling liquid, such as wine.

[0054] In one embodiment, the use of pulsed electromagnetic fields according to the present invention provides increased productivity in the production of any one or any combination of the following products: biofuels, cultures of genetically modified cells and organisms, insulin, monoclonal antibodies, growth hormone, interferon, interleukins, blood factor VIIa, blood factor VIII, blood factor IX, erythropoietin, gonadotrophin, glucagon, vaccine antigen sequences, and mammalian cell cultures.

[0055] Thus, according to the present invention, conventional reactor conditions and equipment may continue to be used for product formation with the addition of generating a pulsed electromagnetic field to create the environment in which the product is located.

[0056] Generally, microbial organisms are electromagnetic systems and respond to electromagnetic changes.

[0057] Typically, the culture is irradiated with PEMF using a device comprising a radio or microwave transmitter positioned so that its use is non-invasive and therefore does not alter any nutritional or formula components of the product.

[0058] In one embodiment, the radiation frequency is preferably 2.4 GHz.

[0059] Typically, the duration of the pulses is in the range of 0.5-1.5 milliseconds, more preferably 1 millisecond. Typically, the pulses are separated by rest periods, which in one embodiment are in the range of 40-60 milliseconds, and more preferably 50 milliseconds.

[0060] Providing a rest period between pulses ensures that the microorganisms are not overwhelmed by the electromagnetic energy, but rather are encouraged to increase metabolic processes and enhance growth rates. This has been found to result in increased metabolite expression and more efficient conversion of nutrients to products, thereby increasing yield and / or reducing the production time required to achieve desired results. Furthermore, the rest period between pulses allows the PEMF-induced activity in the product to relax, thus promoting homogeneity as clusters break apart and naturally form a thermodynamically favorable, open structure for the product.

[0061] In one embodiment, to detect the generation of PEMF, an electronic magnetic field detector may be utilized in the vicinity of the device.The change in product state may be one or a combination of a characteristic and / or a product process.

[0062] In one embodiment, the control means allow the frequency and the numerical sequence of the emitted electromagnetic field to correspond to the dielectric and / or other properties of the product then held in the container.

[0063] In one embodiment, the generated electromagnetic field is pulsed at 2.4 GHz and the system provides low field, typically milliwatt, energy pulses. The circuit is typically programmed to provide one millisecond pulses at 2.4 GHz at a low pulse frequency between 10 and 20 Hz, so that the duty cycle is typically in the range of 1-2%.

[0064] In one embodiment, the duration of the applied electromagnetic field pulse is in the range of 30 minutes to 2 hours and, if desired, can be performed simultaneously with another function, such as cooling the liquid. It will also be understood that the duration of the applied electromagnetic field pulse depends on the type and / or amount of liquid being treated.

[0065] The frequency range in which PEMF is generated is considered the industrial scientific and medical band and is generally characterized as being provided as low fields or low energies in the milliwatt range with short pulse widths of approximately 1 millisecond and with a low frequency pulse rate of typically 15 Hz.

[0066] Typically, this form of electromagnetic field is used in services such as smartphones using Bluetooth and can be modified to pass these frequencies.

[0067] Thus, the present invention provides significant improvements in reducing the time it takes for a component to enter the exponential phase, thereby allowing for a reduction in the overall cycle time for processing of fermentation components. For example, in the production of bioethanol, the improvements detailed herein make it possible to significantly reduce production time, thereby increasing the yield and output of the same equipment conventionally used.

[0068] In one embodiment, the use of PEMF is controlled for use under aerobic conditions, and in one embodiment, the application of PEMF to a material in an aerobic environment is provided. In another embodiment, the method is used to produce butanol from E. coli to form a biofuel.

[0069] Thus, according to the present invention, the use of the methods and apparatus described herein to provide pulsed electromagnetic fields allows for improved and enhanced production yields without adversely affecting the quality of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Specific embodiments of the present invention will now be described with reference to the accompanying drawings;

[0071] Figure 1 An apparatus used according to one embodiment of the present invention is schematically shown.

[0072] Figure 2 The manner in which a device for emitting a pulsed electromagnetic field is used in combination with a product culture medium to be treated is shown.

[0073] Figure 3 An alternative arrangement of the device relative to the product medium is shown.

[0074] Figure 4 The device is shown in use in combination with a container in which the product culture to be treated is contained.

[0075] Figure 5 Shown Figure 4 Another embodiment of the arrangement;

[0076] Figure 6 Shown is how one or more devices may be used as part of a circulation apparatus.

[0077] Figure 7 An apparatus according to one embodiment of the present invention is shown, and components of a control device for use therewith are shown.

[0078] Figure 8 A device according to one embodiment of the invention is shown mounted on a bottle.

[0079] Figure 9 An alternative form of device is shown by which a bottle can be positioned.

[0080] Figure 10 and 11 An alternative embodiment of the device allowing engagement with the bottle is shown;

[0081] Figure 12 An alternative embodiment of the device is shown;

[0082] Figure 13 shows the device in combination with another item; and

[0083] Figures 14a-14c A further embodiment of the device according to the invention is shown, which can be used in conjunction with a container in the form of a cup.

[0084] Figure 15 A graphical representation of a comparison of optical density versus time between a control amount of E. coli and E. coli that has been treated with PEMF;

[0085] Figure 16 Shown is a comparison of dry cell weight over time between a control amount of E. coli and E. coli treated with PEMF.

[0086] Figure 17 The amount of E. coli as a control and the amount of E. coli that have been treated with PEMF according to the present invention are graphically shown as a function of pH over time;

[0087] Figure 18a Shown are changes in cellular respiration over time for control and PEMF-treated E. coli.

[0088] Figure 18b Acid production in E. coli is shown;

[0089] Figure 18c Metabolic intermediates in E. coli are shown;

[0090] Figure 19a and19b Graphically shown are comparisons of control amounts of S. cerevisiae and the same material treated with PEMF with respect to optical density and DCW, respectively;

[0091] Figure 20 A graphical comparison of pH changes over time for a control amount of S. cerevisiae and the same material treated with PEMF is shown;

[0092] Figure 21 A graphical representation of the changes in cellular respiration over time for a control amount of S. cerevisiae and the same material treated with PEMF is shown.

[0093] Figure 22 and 23a and 23b show metabolic activity in Saccharomyces cerevisiae;

[0094] Figure 24 Metabolic intermediates in Saccharomyces cerevisiae are shown;

[0095] Figure 25 Ethanol production in Saccharomyces cerevisiae is shown;

[0096] Figure 26 One embodiment of an apparatus for introducing a pulsed electromagnetic field into a container in which a product to be treated is located is shown;

[0097] Figure 27 and 28 Shown Figure 26 potential use of the device together with the container;

[0098] Figure 29 Another embodiment of the device according to the invention is shown;

[0099] Figures 30-35 The present invention relates to mammalian cell culture test results according to one embodiment of the present invention. DETAILED DESCRIPTION

[0100] First reference Figure 26 , shows one embodiment of an apparatus that can be used to introduce a pulsed electromagnetic field into a container in which a product to be treated is located.

[0101] In one embodiment, the probe 1 is provided with an outer housing 3, such as a glass tube, which in one embodiment has a sealing cap 5 fixed at one end to a glass shell 7 and has a suitable attachment structure, typically including a flange that allows an airtight seal to be formed, which allows the glass tube to be attached to the wall or another component of the container into which the probe 1 is to be inserted, thereby mounting the probe in a fixed position. The main part of the probe, typically the glass shell 1, will be located within the container. Within the shell 3, a printed circuit board 7 is provided, which has parallel circuit traces for modular power supply and series circuit traces for module data feedback and programming input. The circuit board also includes a rechargeable battery, so it will be understood that the printed circuit board serves as a means for powering the module 9 and providing control data to and from the module in order to operate it in the desired manner.

[0102] A series of spaced modules 9 are positioned in the desired configuration for a particular application and, in this case, are equally spaced along the length of the housing 3. Each module is capable of emitting a pulsed frequency that passes through the housing wall and into the container, thereby acting on the product held therein. The arrangement of the modules 9 on the wick support 11 allows for proper spacing of the modules 9 from the walls of the housing 3 and, therefore, provides a degree of thermal insulation from heat generated by other processes within the container, such as a sterilization process, which may allow the housing to be sterilized by steam and, in one embodiment, the wick 11 may be removed from the housing during this process and then reinserted into the housing.

[0103] Figure 27 and 28 The potential different uses of the probe 1 according to the invention are shown and Figure 27 , a probe 1 is shown introduced into the interior of a container 13 from the top of the container so that the probe is centrally and axially positioned in the container to allow electromagnetic field pulses to be emitted therefrom 360 degrees around the probe and thereby provide substantially uniform treatment of the product within the container.

[0104] Figure 28 A configuration is shown in which multiple probes 1 are positioned through various openings in the container wall, and in this embodiment, the probes 1 extend horizontally into the container 13 and are offset 90 degrees so as to provide an electromagnetic field pulse from each probe. It is envisioned that this and other multiple probe configurations may be suitable for use in larger capacity containers and / or with products contained within the container that benefit from more intense exposure to the electromagnetic field.

[0105] Figure 29Another embodiment of the apparatus is shown, in which a container 13 is again provided, containing the product to be processed therein. In this embodiment, a sleeve 15 is provided, which is cylindrical and has a plurality of modules 9 positioned in a selected matrix configuration for emitting pulsed electromagnetic fields therefrom, and a control module 7 connected to each module, typically by wires integrally positioned with the cylindrical material, to allow power and control data to be sent to and received from the modules 9. In the embodiment shown, the sleeve 15 can be moved as indicated by arrow 7 to be positioned around the container, or in another embodiment, and particularly for use with containers that are repeatedly used for the same purpose, the sleeve can be provided as an integral part of the container's wall structure, or the modules can be arranged in the desired matrix configuration and positioned with the wall structure without the need for a supporting sleeve.

[0106] Now refer to Figure 1 Another form of apparatus according to the present invention is provided, and in this embodiment, the apparatus is configured to treat a live culture of a microbial system, typically provided as part of a product, and to increase its productivity. This is achieved by exposing the culture to a relatively low energy pulsed electromagnetic field (PEMF), typically in the order of microwatts per liter of culture medium, the frequency of which may be in the microwave range, and the pulses at low frequencies, such as 10 to 200 Hz. This method step has been found to increase the growth rate and expression levels of the microbial culture.

[0107] In one embodiment, PEMF transmission can come from one or more modules that include a control device and a transmitter similar to those used in a Personal Area Network system and can be controlled to allow PEMF to be generated from the module and powered by batteries or directly from a mains supply. Figure 1 An example of the components required to generate PEMF from a module 2 is shown, which includes a power source 4, a data processor 6, a memory 8, a signal generator 10 and an antenna 12.

[0108] The module 2 can be placed under or against a microbial culture 16, such as a fermentation of yeast in a sugar-based medium to produce alcohol and carbon dioxide. The fermenter can be exposed from any direction because the signal generated is omnidirectional and does not depend on location but only on the walls of the nearby, preferably contacting, fermentation vessel.

[0109] Figure 2One possible arrangement is shown in which the module 2 is located below a vessel 14 in which a culture medium 16 is placed, so that the electromagnetic field pulses move upwards through the culture as shown by arrow 17. However, because the module is omnidirectional in the direction of emission of the pulsed electromagnetic field, other arrangements are possible, e.g. Figure 3 In the illustrated form, the module 2 is mounted to a side 18 of the vessel 14 so that PEMF can be applied in the direction indicated by arrow 20 .

[0110] In another embodiment, multiple fermentation or bioreactor vessels can be processed simultaneously, such as Figure 4 and 5 For example, in Figure 4 1 shows several containers 22 in a top view, which in this example contain a product in the form of a fermented alcoholic beverage. The beverage may be sparkling wine or unpasteurized bottled beer, and the containers, such as bottles 22, are positioned around the module 2 as shown so that each bottle 22 is equidistant from the PEMF antenna 12 of the module 2 and so that the fermented components of the product 16 in each bottle receive the same exposure to the PEMF.

[0111] exist Figure 5 , another embodiment of a plurality of containers 22 of fermenting microbial cultures exposed to PEMF from an array of modules 2 is shown. In this embodiment, a plurality of containers 22 are simultaneously exposed to PEMF in an array formed such that module 2' serves a group of four containers 24, a second module 2" serves a group of containers 26, module 2'" serves a group of containers 28, and module 2"" serves a group of containers 30. It will be appreciated that the array can be organized so that it is easily transported and assembled during storage so that the containers and the cultures contained therein can be exposed for a set period of time before being moved to a new group of containers for exposure.

[0112] For large-scale production of PEMF for fermentation or cell culture, the modules 2 can be housed in a waterproof, sterile support in the form of a housing that is transparent to the electromagnetic frequencies employed. The PEMF modules 2 thus described can be placed around the vessel wall 24 to provide an extensive array of the modules that emit PEMF into the culture medium 16 within the container vessel as indicated by arrows 26. Figure 6 As shown. In another embodiment, the module can be placed within a support in the form of a circulating side arm, so that the product flows through the module and receives PEMF irradiation as it does so. These embodiments are particularly suitable for large-scale microbial cultivation. It is assumed that the PEMF treatment described generally herein provides electromagnetic interference to the charged surfaces within the living cells, which stimulates increased growth and / or metabolite expression.

[0113] In the case of bottled sparkling wine, it can be inferred that the increased CO2 production improves the mousse and texture of the sparkling wine in the mouth. In other types of products that include alcohol, alcohol activation can increase productivity, as yeast is stimulated and encouraged by PEMF to produce more alcohol (primarily ethanol). In this case, the alcohol can be the alcohol contained in fermented beverage products (such as wine and beer) or fermented mash before distillation.

[0114] Increasing productivity by the methods described herein to provide additional and / or higher productivity of alcohol in the production of biofuels is also described as a use of the present invention.

[0115] exist Figure 7 , a device 102 is shown having a housing 110 with components that allow the device to be used to generate an electromagnetic field in the form of pulses in a liquid 112 contained in a bottle. The device components include a power source, in this case a battery 114, which is located within the housing 110. The battery can be recharged or replaced when expired.

[0116] A switch 116 is provided to allow the device to be turned on and off, and a visual display 118 may be provided in the form of simply indicating the operation of the device and / or supplying power to the device 102, or in other forms, wherein the display provides a decorative effect in addition to its functional effect, for example being arranged to display the logo of a company, which may be, for example, the manufacturer of the liquid in the bottle used with the device.

[0117] Another indication is provided in the form of light source 120 which may illuminate once a sufficient period of electromagnetic field emission has elapsed for a quantity of liquid to be effectively treated, thereby indicating that the device has ceased use and that the liquid 112 has been conditioned using the electromagnetic field for a sufficient period of time.

[0118] A timing device 112 may be provided that allows the user to select a specific operating time for the device in conjunction with the bottle and the liquid contained therein. It will be understood that, in addition to the components described above, electrical control circuitry and components are provided within the housing 110 for controlling the pulse emission and generation of the electromagnetic field in the desired form, and that the housing wall 122 is configured to be effectively transparent to the electromagnetic field so as to allow the electromagnetic field to pass through and into the container 4 when the container 4 is located therein.

[0119] Figure 8One embodiment of a means by which the housing 110 may be positioned together with the bottle 104 is shown, in this case by providing a sleeve 124 shown in cross-section which passes around the bottle 104 and the device housing 110 at a junction 126 therebetween and engages them together.

[0120] exist Figure 9 , another embodiment of an engagement means is shown in the form of a strap 128 positioned together with a collar 130, the collar encircling the neck 108 of the bottle, with the strap 128 extending from the device housing to the collar and thereby holding the device housing 110 in contact or abutment with the base 130 of the bottle at the abutment 126. Typically, the strap is configured to be resilient and thereby bias the device 102 towards the bottle base 130.

[0121] exist Figure 10 , there is shown the manner in which the device housing may be shaped so as to form, in this embodiment, a surface 122 having a protrusion 132 which is shaped to seat in a recess 134 in the base 130 of the bottle, as shown. Figure 7 shown.

[0122] Alternatively, as Figure 11 As shown, the positioning means of the device may simply be a flat portion 122 on the housing 110, with the container 104 being placed on the flat portion 122 and being freestanding.

[0123] In other embodiments, the device may be incorporated into items having other functions and Figure 12 In FIG. 1 , a cooling sleeve 136 is shown which has cooling means therein to allow the bottle 104 to be cooled when it is placed in the cavity 138. Then, according to the present invention, in addition to the cooling means, a further cooling means is provided. Figure 1 Components are described that are disposed in the base 140 and / or sidewall 142 to emit an electromagnetic field from the sleeve and into the liquid 112 held in the bottle 104 .

[0124] exist Figure 13 , an ice bucket 142 is shown having a cavity 144 for receiving ice and water 146 therein, and further, the base 150 and / or walls 148 of the ice bucket are provided with appropriate circuitry to allow an electromagnetic field to be generated from the circuitry and into the liquid in the bottle 104 then held in the cavity.

[0125] Now turn Figures 14a-14d, a container in the form of a cup 152 is shown having a base 154, a neck 156 and a cavity portion 158 for holding liquid therein. Again, the device housing 110, which in this case includes a sleeve 160 or collar 162 as shown, can be provided in a variety of different forms. Generally, the same components will be included in the device regardless of the particular type of container with which the device is to be used, and in one embodiment, lighting means 164 provide both a functional effect and also as a visual decorative effect on the portion of the cup, such as Figure 14b shown.

[0126] Now provide as this article about Figures 1 to 14d Specific examples of uses of the described apparatus and methods;

[0127] Example 1 - Growth rates of yeast in a typical home wine fermentation.

[0128] The experiment took place in Haddenham, Buckinghamshire, from May 7, 2018, to May 14, 2018.

[0129] Two commercial wine kits were obtained and prepared and started identically in 5-liter demijohns. The supplied yeast was added and the two cultures were separated by more than 30 feet. A smartphone was placed against the cup of one demijohn (active sample). The smartphone, a Galaxy S4, was loaded with an application that controlled a personal area network, PAN, microwave system (trade name Bluetooth), which transmitted a pulsed electromagnetic field with the characteristics detailed below. The other demijohn underwent irradiation and fermented normally (control sample).

[0130] Irradiation process

[0131] A smartphone (with the professional app in active mode) was placed against the outside of the active wine bottle. The control app was used to control the PAN and provided 2.4 GHz at a 15 Hz pulse rate in 1 millisecond pulses. The smartphone was in active mode for 2 hours. This was repeated every 12 hours (this resulted in 2 hours of active pulse exposure) for one week.

[0132] The bottles were observed regularly and it was seen that carbon dioxide production (as evidenced by the rate of bubbles through the air lock system) in the active bottles was on average more than double that of the controls on day 3.

[0133] Bubble generation rate on day 3

[0134] Active wine bottle: 9 bubbles per minute

[0135] Control wine bottle: 4 bubbles per minute

[0136] At the end of the week, the lees (spent yeast cells) were observed and compared to the control storage bottles. It was seen that significant additional growth had occurred in the active samples, as evidenced by the depth of the lees.

[0137] Active sample: Depth of lees 180 mm

[0138] Control sample: lees depth 80 mm

[0139] Yeast growth increased by 225%, indicating enhanced metabolism and therefore increased alcohol and carbon dioxide production rates (as evidenced by bubble production rates above)

[0140] Example 2: Live, Bottled-Conditioned Beer

[0141] The experiment was conducted over 2 days from May 10, 2018 to May 12, 2018 in Haddenham, Buckinghamshire

[0142] The sample was 500ml of St Austell Brewery's 'Proper Job' India Pale Ale, a live bottle beer that has not been pasteurised and therefore retains live yeast in the bottle that can respond to pulsed electromagnetic fields.

[0143] The two bottles of the aforementioned beer were purchased from the same shelf at Waitrose in Thame, Oxfordshire, and then separated by at least 30 feet at the same premises in Haddenham to ensure that only the active sample could receive PEMF from the smartphone.

[0144] A Galaxy S4 smartphone purchased from the Google Play Store and installed with the corresponding app was placed against a bottle of live beer, with the app activated in active mode. This is an active sample.

[0145] The active samples were treated as above for two hours, twice a day, for a total of 2 days.

[0146] Another bottle of the same beer, untreated (i.e., without PEMF), was held at least 30 feet from the smartphone. This served as the control sample.

[0147] After 2 days, the bottles were opened and poured into beer glasses and their characteristics were observed. The two beers were then tasted and their sensory differences were evaluated.

[0148] Active beer: It can be seen that the foaming is significantly greater, and the pour must be interrupted to allow the foam to settle. While in the glass, the gas foam on the top of the beer lasted for 8 minutes, and bubbles were seen rising for 20 minutes. Compared to the control, the color of the beer was 2 shades (2 degrees) darker than amber.

[0149] Upon tasting, it was clear that the beer had a creamy, smooth texture and was very aromatic compared to the control. The length and finish of the beer was also noticeably different from the control, see below.

[0150] Control beer: The beer can be poured continuously, and the foam remains in the cup throughout. The foam head disappears quickly within 2.5 minutes, and the bubbles stop within 4 minutes.

[0151] When tasted, the control beer lacked the creamy and mellow texture of the active sample and had little effect on the fizzy, mouth-delivering flavor. The finish was short and lacked flavor definition compared to the active sample.

[0152] Example 3: Sparkling Wine (Cava and Champagne)

[0153] The experiment took place from May 14, 2018, to May 21, 2018, in Haddenham, Buckinghamshire.

[0154] I picked up 3 pairs of sparkling wines from Waitrose in Thame, Oxfordshire. 1 pair of Bollinger Champagne, 1 pair of GH Mumm Cordon Rouge Champagne and 1 pair of Waitrose own label Cava.

[0155] The pairs were separated, one of which became the active specimen, and the other part of each identical pair was separated by at least 30 feet.

[0156] The three active bottles were placed in the middle of a group of three, along with a Galaxy S4 smartphone, so that each bottle was equidistant from the smartphone. Prior to the experiment, the smartphone was loaded with an appropriate app. Once activated, the app controlled the PAN system, sending 1-millisecond 2.4 GHz pulses at a 15 Hz pulse rate.

[0157] As described above, a smartphone with the app activated was placed between active samples for 2 hours, twice daily for the duration of the experiment. After 7 days of treatment, the bottles were combined with their control counterparts and refrigerated before tasting.

[0158] Tastings of the sparkling wines were carried out by Head Noses at the tasting laboratory of Corney and Barrow wine importers at their premises at 1 Thomas Moore Street, London.

[0159] Tasting Results: Each sparkling wine was opened normally, but it was noted that in each case the treated sample had a louder, lower frequency "boomy" pop upon opening.

[0160] The treated samples produced more bubbles in the cup and the bubbles stayed in the cup significantly longer than the control. Every "nose" commented that there was a significant improvement over the "mouse," which was described as smooth like silk.

[0161] Each of the 'tasters' commented that the overall quality of the drinking experience was improved in each case of the Bollinger, Mumm and Waitrose Cava.

[0162] In other embodiments, the above described array of modules 9, 2 outside and inside a container containing a product therein may selectively result in increased productivity in the fermentation of industrial products such as Aspergillus production (eg citric acid for the beverage industry).

[0163] In another embodiment, the product cell culture or fermentation product receiving PEMF is a genetically modified organism. In this case, the desired metabolite can be insulin from modified yeast or other biopharmaceutical proteins (e.g., monoclonal antibodies), other hormones (e.g., glucagon), growth hormones, gonadotropins, hematopoietic factors (e.g., erythropoietin), or colony-stimulating factors. Proteins that can also increase production or productivity include, but are not limited to, interferons, interleukins, and blood factors (e.g., Factor VIIa, Factor VIII, and Factor IX). Additionally, thrombolytic agents produced by cell culture include tissue plasminogen factors. Furthermore, other biopharmaceutical products whose productivity can be increased according to the present invention are vaccines, such as hepatitis B or influenza antigens.

[0164] The electromagnetic modulation can have different frequencies and waveform shapes. There may also be many pulse frequencies sufficient to stimulate the growth of fermentation and cultured microbial systems.

[0165] In practice, the use of electromagnetic frequencies is subject to legal constraints. The frequency band around 2.4 GHz was chosen because it is believed to provide a good balance between the modulated electric field and the dielectric properties of water, causing water to rotate in the presence of 2.4 GHz. Frequencies at and near 2.4 GHz are also license-free and have been reserved by international governments for use by industry, science, and medicine. This is known as the ISM band.

[0166] In various embodiments, the duration of the pulses, the pulse frequency, and the electromagnetic frequency can vary depending on the product culture being treated, and the duration of treatment can vary from hours to days or weeks, and exposure to PEMF can be continuous or given periodically.

[0167] Now turn Figure 15-18c , which graphically shows the results of a comparison between a control amount of E. coli and E. coli that has been treated using PEMF according to one embodiment of the present invention.

[0168] First reference Figure 15 , the graphic legend shows that although the optical density measurements at 600 nm did not show any significant differences between the control fermentations and the materials that had been treated with PEMF, Figure 16 The final cell concentration in grams per liter (g / L) of dry cell weight achieved after 24 hours of incubation is shown, with a 57% increase for E. coli exposed to the PEMF material but not the control amount.

[0169] Figure 17 Regarding pH, although the initial pH of the control E. coli was 6.68, which was different from the pH of 7.59 for the PEMF-treated E. coli, the pH was controlled at 7 in both experiments, and the acidification activity of the culture in the PEMF fermentation was much stronger than that of the E. coli that was not exposed to PEMF. Although the control system could not adjust the pH quickly enough to reduce the pH to 6.20 (relative to 6.58 for the E. coli control), this may mean that PEMF has led to higher production of organic acids.

[0170] Then, about Figure 18a, relative to a certain amount of material that had been exposed to PEMF, there was an overall higher metabolic activity, as the CO2 produced and released into the culture medium was significantly higher by 74% than in the control portion of E. coli. Therefore, this means that cellular respiration is more important under PEMF conditions, and, as a result, the production of secondary metabolites (such as formate and acetate) occurs, which may explain the more dramatic drop in pH observed.

[0171] Figure 18b The total amount of base transferred to the fermenter to raise the pH was shown to be greater for the PEMF-exposed material than for the control, indicating that the bacteria produced more total acid under PEMF conditions.

[0172] Then, Figure 18c The results show that for the control E. coli material, an unknown metabolic intermediate was produced throughout the log phase of growth, which had already entered the post-stationary phase. However, for the material exposed to PEMF, this intermediate was consumed only after a brief appearance in the log phase. This suggests that fermentation under PEMF conditions does not require short-term energy storage in the form of intermediates.

[0173] E. coli, a strain known for its ability to produce two organic acids (acetic and formic), apparently expressed a higher percentage of these compounds, approximately 15%. This difference was statistically significant. Clearly, if this increased expression is replicated in strains engineered for pharmaceutical production, it could yield beneficial results.

[0174] Now refer to Figures 19a-25 , and Figure 15-18c The same test was conducted, but in this case on a material made from the yeast S. cerevisiae. Regarding these results, the material exposed to PEMF grew faster than the control and also entered the exponential growth phase earlier than the control. After a 5-hour lag phase, the fermentation exposed to PEMF entered the exponential phase after 6 hours of incubation. In fact, higher OD and DCW values were recorded from this point on. While the final cell concentrations of the control and PEMF-exposed materials were equivalent after 25 hours of incubation, and this may mean that the fermentation of the control was eventually caught up by the material under PEMF stimulation, these results do indicate that faster production (throughput) can be achieved using PEMF exposure compared to the material not exposed to PEMF, allowing for at least the same level of fermentation to be achieved more quickly.

[0175] Regarding pH value, Figure 20As shown, in the PEMF fermentation, the acidification activity of the culture began between 30 minutes and 1 hour earlier than in the control material, thus also having the potential advantage of faster fermentation production.

[0176] about Figure 21 As for cellular respiration, CEO transfer ratio (CTR) showed that although the overall maximum cellular respiration was reached faster under PEMF stimulation, which may be due to the cells entering the exponential growth phase earlier, there was no significant difference in overall cellular respiration. Figures 19a-21 , showing that faster throughput rates can be achieved without adversely affecting the ultimate level of fermentation achieved. As a result, in a single use, the yeast will produce greater amounts of ethanol in a shorter period of time, thereby increasing productivity on a commercial and industrial basis.

[0177] Figure 22 、 23a Figures 23b and 23b show that under PEMF conditions, the growth, respiration, and acid production rates of Saccharomyces cerevisiae were faster than those under control conditions. This indicates that Saccharomyces cerevisiae reached the logarithmic phase and thus the production phase earlier under PEMF conditions.

[0178] The significant differences in fermentation achieved with PEMF treatment showed that higher concentrations of alcohol could be produced, and it is believed that, in some cases, maximum alcohol production was reached earlier in the fermentation process than in materials not exposed to PEMF.

[0179] about Figure 24 , demonstrating that for control materials and conditions, S. cerevisiae cultures contained a fairly constant amount of an unidentified metabolic intermediate throughout fermentation. However, for materials exposed to PEMF, this intermediate completely disappeared during the early log phase, only to reappear in the late log phase, suggesting that it was somehow being used up and then regenerated.

[0180] about Figure 25 , showing that the material was exposed to PEMF, and that the same organism produced ethanol much later in the fermentation than the control material. However, despite the delay, the concentration of ethanol in the later stages of fermentation was higher in the material exposed to PEMF, indicating that the use of PEMF had a substantial impact on alcohol production by this strain.

[0181] It should be understood that although the results are from tests conducted on E. coli and S. cerevisiae, other cultures, such as mammalian cell cultures, can be used. Since mammalian cell cultures are completely aerobic, it is believed that the results from these cultures will be as inventive and novel as the disclosed results.

[0182] Overall, the test results showed surprisingly beneficial data for E. coli, with the PEFM system producing a 57% increase in culture weight and a 74% increase in metabolic activity in E. coli compared to the control. Regarding yeast, the PEFM-treated material entered the exponential phase much earlier, potentially reducing overall batch cycle times in bioethanol production.

[0183] In this embodiment, the apparatus and method according to the present invention are particularly effective under aerobic conditions, which is the state of the yeast before it begins to produce ethanol and the E. coli under constant aerobic conditions. Since many biopharmaceuticals are expressed in E. coli, this has potential major advantages for pharmaceutical production.

[0184] Thus, the use of PEMF technology increased metabolic activity in E. coli, increased the rate of ethanol production in Sacchoromyces cerevisiae, and favorably affected the production of metabolic intermediates in both E. coli and S. cerevisiae.

[0185] Another embodiment and example of the use of the present invention is the use of pulsed electromagnetic field (PEMF) modalities in mammalian cell culture. According to the present invention, PEMF technology was used in conjunction with a glass stirred tank bioreactor to produce IgG subclass 2 (IgG2) from an IgG-expressing hybridoma cell line that had previously been grown in conventional cell culture flasks and in a STR, with IgG yields in the range of 30-50 μg / mL and 130 μg / mL after dialysis and concentration, respectively.

[0186] The aim of the test was to evaluate whether PEMF has an effect on mammalian cell metabolism, particularly in terms of increasing IgG production, and to assess whether cultivation in STR of an IgG-expressing cell line would result in competitive IgG production (targeting 300-500 μg / mL). Two independent experiments were performed:

[0187] 1. The purpose of the first experiment was to perform a quadruple benchtop 1L cell culture grown in the absence of any ambient PEMF (negative control experiment). During this experiment, a set of parameters for growing the cell line in an STR was determined using a literature review, equipment supplier recommendations, test runs, and internal knowledge of the cell line.

[0188] 2. The second experiment aimed to perform a quadruple benchtop 1 L cell culture grown in the presence of PEMF (test experiment). During this experiment, the same set of parameters as those predetermined in aim 1 were used again, without taking into account the yields previously achieved.

[0189] In the scenario, use the following parameters:

[0190] 1. pH control using 7.5% sodium bicarbonate and CO2 to maintain pH between 7 and 8.

[0191] 2. The gas flow rate was set to 3 L / h to minimize the flow rate deviation of the bioreactor and improve the reproducibility of the experiment.

[0192] 3. Implement four PEMF modules, each directly attached to one of the four bioreactors during PEMF operation.

[0193] 4. Use air for DO control to maintain a minimum dissolved oxygen concentration of 40%.

[0194] In the test, the PEMF device was configured as four modules that emitted unique PEMF patterns. For the control condition, no PEMF device was used, and all other PEMF / Bluetooth devices were turned off and removed from the laboratory throughout the cell culture process. For the experimental condition, each of the four PEMF device modules was placed in direct contact with one of the glass stirred tank bioreactors (STR), turned on, and remained on throughout the cell culture, while all other PEMF / Bluetooth devices were kept outside the laboratory. Cell cultures were monitored using an online gas analyzer, online and offline pH monitoring, offline cell count measurements, and HPLC analysis of IgG production. Mouse hybridoma cell lines and culture media were premixed in 1 L sterile bottles provided by The Antibody Company. The culture media included GlutaMAX TM Dulbecco's modified Eagle's medium (DMEM, Life Technologies) and IgG-reduced fetal bovine serum (FBS) (Life Technologies) were added. Pluronic F-68 (Life Technologies) was added at a dilution of 1:100 to reduce foaming in the reactor. For more information on medium components, see the Appendix.

[0195] Before each cell culture, the DASGIP reactor was autoclaved the day before incubation. The reactor was stored overnight in a laminar flow hood and periodically treated with UV light to maintain sterility.

[0196] Control runs: A preculture of mouse hybridoma cells was prepared by The Antibody Company and divided into 4 x 1 L flasks of culture medium (as described above) at a concentration of 3.5 x 10 5cells / mL, with a cell viability of 64.8%.

[0197] PEMF run: A pre-culture of murine hybridoma cells was split into 4 x 1 L bottles of culture medium (as described above) at a concentration of 4.87 x 10 5 cells / mL with a cell viability of 77%.

[0198] These were immediately transported to FlexBio where 1 L of preculture was placed into each reactor of an Eppendorf DASGIP parallel bioreactor system equipped with a pitched blade impeller.

[0199] Table 1. Control conditions used in the four reactors in each run.

[0200]

[0201]

[0202] 7 mL samples were extracted from each fermentor at the sampling points specified in Table 2.

[0203] Table 2. Time from the end of incubation to the start of each sampling point discussed in this report. Throughout the Results section, sampling times are rounded to the nearest date.

[0204]

[0205] For cell culture using Pulsar Technology, four PEMF devices were set up and turned on by attaching one device to each of four glass DASGIP reactors. They remained turned on and plugged in throughout the cell culture run. Unfortunately, one bioreactor was terminated due to a malfunction in the operational setup.

[0206] The following parameters were analyzed:

[0207] Off-line pH was measured using a HANNA HI8424 pH meter (Hanna Instruments).

[0208] 2 mL of sample was transferred to a 15 mL falcon tube and the probe was inserted into the tube below the liquid line. This was done within 2 minutes of removing the sample from the reactor to reduce rapid CO2 outgassing, which would affect the pH of the sample.

[0209] Growth rate was measured by transferring 50 μL of the remaining unfiltered sample into an Eppendorf containing 50 μL of Trypan Blue (Sigma Aldrich). The sample was mixed with the stain by gently pipetting up and down. The stained sample was applied to a cell counting slide (Nexcelom Bioscience) and cells were counted using an automated NexcelomCellometer (Nexcelom Bioscience).

[0210] Through this process, the following information is recorded:

[0211] Total cell count (cells / mL)

[0212] Viable cell count (cells / mL)

[0213] Live / dead cell ratio

[0214] Average cell diameter (μm)

[0215] ·active(%)

[0216] Blood glucose concentration was measured by using the remaining sample filtered at 0.22 μm to remove all cells and using a commercially available blood glucose meter (Accu-Chek Mobile) according to the manufacturer's instructions, wherein 10 μL of the filtered sample was transferred to a test strip and the displayed value was recorded.

[0217] The concentration of monoclonal antibodies was measured by storing a certain amount of sample at -20°C and then thawing under sterile conditions.

[0218] To determine IgG concentration, supernatant samples were analyzed using ion exclusion chromatography.

[0219] HPLC: Agilent 1290 Infinity.

[0220] Analytical column: Thermofisher Scientific Poros A20.

[0221] Buffer A: 50 mM phosphate, 150 mM NaCl.

[0222] Buffer B: 12 mM HCl, 150 mM NaCl.

[0223] Elution: Gradient elution (Table 3)

[0224] Injection volume: 20 μL.

[0225] Measurement: Absorbance at 280 nm and 214 nm.

[0226] Standard 2: Normal mouse IgG (Sigma Aldrich, 12-371) (Generate standard curve)

[0227] Table 3. Summary of gradient elution for IgG analysis.

[0228] Time (minutes) ％B Flow rate (mL / min) 0.00 0 2.5 4.50 0 2.5 4.51 100 2.5 7.50 100 2.5 7.51 0 2.5 15.00 0 2.5

[0229] Statistical analysis

[0230] Microsoft Excel (2016) was used for graph generation, data distribution, and statistical analysis. The Student t-test was used to analyze the experimental comparison of independent sample data. Statistical significance was considered if P < 0.05. All statistical analyses were performed using data from four independent biological replicates for the control group (n = 4) and three independent biological replicates for the treatment group (n = 3). Error bars describe the standard deviation of the sample groups.

[0231] Analysis of the results showed that the mean total cell number of cell cultures exposed to PEMF was higher in terms of growth rate and metabolic activity than that recorded when cells were cultured without the PEMF device (control cultures) ( Figure 30 After 4 days of growth, there was a significant difference between the PEMF-treated and untreated cells (p=0.048) ( Figure 30 After 6 days of incubation, the control bioreactor reached a maximum average total cell count of 1.44 x 10 6 cells / mL, while cultures exposed to PEMF achieved an average total cell count of 6.06 x 10 6 cells / mL( Figure 30 After 4 days of growth, the mean total cell number in treated cultures was still higher than that in controls, but no significant difference was recorded (p>0.35) ( Figure 30 ).

[0232] From day 0 to day 2, the mean total number of viable cells in cultures exposed to PEMF was higher than in control cultures, although this difference was not significant (p>0.122). After 3 days of growth, the number of viable cells was significantly higher in PEMF-treated cultures compared to control cultures (p=0.009) ( Figure 30 After 3 days of growth, the average total viable cell number decreased in cultures exposed to PEMF, while the number of viable cells in control cultures continued to increase slightly ( Figure 30 ).

[0233] In both runs, glucose consumption followed the same pattern, with rapid depletion from day 1 to day 3 after incubation and then remaining relatively constant until harvest ( Figure 31 Although not considered significant (p>0.05), glucose concentrations in PEMF-treated cells were consistently lower than those in control cultures from day 1 to day 3 ( Figure 31 ).

[0234] As discussed above, the lower glucose concentrations in the culture medium of PEMF-treated cells compared to control cultures may indicate a higher rate of glucose consumption in this culture, which may be directly related to the higher cell numbers observed in cells exposed to PEMF ( Figure 30 Therefore, we can hypothesize that PEMF induces a higher rate of glucose consumption, which may indicate a higher rate of cellular respiration (necessary for cell division). However, more in-depth studies must be conducted to determine whether this hypothesis is correct.

[0235] Results from a previous study (ECO-410) showed that tighter pH control is essential to counteract the effects of lactic acid production on the acidity of the cell culture medium. In this study, the pH of the culture was controlled using CO2 and 7.5% sodium bicarbonate to ensure that the pH remained in the range of 7.3-7.8 ( Figure 32 ).like Figure 32 As shown, the pH of both PEMF-treated and untreated cultures remained fairly stable throughout the experimental run.

[0236] Oxygen uptake occurred at a slightly faster rate in cells exposed to PEMF relative to controls, with PEMF-exposed cultures reaching their lowest dissolved oxygen percentage (10.5%) in 3 days, compared to 3.5 days for control cultures (6.7%). Figure 33 ). As discussed in previous studies, during aerobic respiration (in the presence of oxygen), glucose is catabolized and dissolved oxygen is taken from the culture medium and used in the electron transport chain to produce ATP. When cells enter their exponential phase, they divide rapidly, producing and utilizing large amounts of ATP. When oxygen levels become too low, cells switch from oxidative phosphorylation to lactic acid fermentation, and cell division ceases while the percentage of dissolved oxygen (DO) rises again. In this study, the consumption of dissolved oxygen was most pronounced for cells exposed to PEMF until day 3, as was glucose consumption and total cell number ( Figure 30 , 31, 33). Thus, overall, the data seem to indicate that PEMF does induce higher cellular metabolic activity.

[0237] Air flow during the cell culture run increased at a higher rate in cultures exposed to PEMF compared to control cultures, reaching a maximum flow rate (3 sL / h) within approximately 2 days of growth (compared to 2.5 days of growth, respectively) ( Figure 33 In both experimental runs, the decrease in the percentage of dissolved oxygen indicated that the cells were taking up oxygen faster than it was being introduced into the culture medium ( Figure 33 ).

[0238] During the entire run, the total gas flow rate was maintained at 3sL / h, allowing more air to be pumped into the system.

[0239] The DASGIP bioreactor system can introduce up to three different gases (or gas components) into each of the four bioreactors for the duration of the experimental run. In this study, the three individual gases were carbon dioxide (CO2), nitrogen (N2), and an air mixture (approximately 21% oxygen, 78% nitrogen, 0.04% CO2). The gas flow rates were set to 3 sL / h, which means that the combined flow rate of all three gases must always equal 3 sL / h. Figure 34 In this study, nitrogen was used as an inert gas to maintain the required gas flow rate ( Figure 34 At the beginning of an experimental run, cells utilize oxygen from the culture medium at a low rate, so the ratio of air to N2 entering the system is fairly equal ( Figure 34 As the run continues and the oxygen demand of the culture increases, the proportion of air in the total gas mixture is increased to the maximum limit (3 sL / h), with a small volume allocated for CO2, for pH control, and almost no N2 ( Figure 34 ). When the cells enter anaerobic respiration, the glucose concentration is depleted and the cells stop dividing and switch to anaerobic fermentation metabolism. As mentioned above, the demand for O2 decreases and results in a decrease in the amount of air entering the system. This can be compensated by increasing the amount of nitrogen to ensure that the total gas flow rate remains close to 3sL / h ( Figure 34 ).

[0240] Base addition started earlier in PEMF-treated cultures compared to control cultures, at approximately 57.5 h (2.5 days) and 68 h (3 days) post-incubation, respectively ( Figure 35 ). More base was added to the PEMF-treated cells compared to the untreated cells, 15.2 mL and 14.2 mL, respectively ( Figure 35The earlier addition and higher total base strongly suggest that cells in the PEMF-exposed cultures produced lactate earlier in the cell culture run and at higher concentrations compared to the control cultures. Consistent with the higher rates of oxygen uptake and glucose consumption, this may indicate that cells in the PEMF-exposed cultures utilized oxygen at a faster rate and entered lactic acid fermentation before cells in the control cultures ( Figure 31 、 33 and 35).

[0241] As mentioned above, PEMF-based metabolism maximizes glucose uptake during the first phase of growth, but lactate production is ultimately maximized under PEMF exposure. Indeed, under electromagnetic field biostimulation in a pH-controlled environment, cells can produce higher concentrations of lactate (a byproduct produced during anaerobic conditions), as seen in Saccharomyces cerevisiae (also a eukaryotic cell), where ethanol production (also a byproduct of anaerobic fermentation) is 20% higher than when cells are exposed to PEMF.

[0242] During the first phase of growth (from incubation to glucose depletion), the total cell density of hybridoma cells in cultures exposed to PEMF was shown to be higher compared to control cultures. During the same period, the number of viable cells was also found to be higher in the PEMF-treated cultures. The glucose metabolic rate was also increased in the PEMF-exposed cells, suggesting that the PEMF device and its use can positively influence the growth and metabolism of murine hybridoma cells by stimulating and maximizing nutrient uptake.

[0243] In both control and PEMF-exposed cells, cells proliferated during exponential growth, resulting in a higher oxygen demand, as observed in decreased DO levels. Specifically, cells exposed to PEMF displayed stronger and faster growth, supported by higher glucose uptake. This led to earlier oxygen-limited conditions than in control cells, thus triggering lactate production earlier. It was also shown that, in a pH 7.4-controlled environment, cells exposed to PEMF were able to produce higher concentrations of lactate (indirectly observed by higher base addition) than control cultures. Lactate inhibits IgG production, and lower IgG levels were recorded in cells exposed to PEMF.

[0244] DO control is crucial for ensuring the highest growth rate for murine hybridoma cell cultures in stirred bioreactors and preventing lactate production. Furthermore, this experiment raises the possibility that PEMF stimulates lactate production, similarly to glucose consumption (since a similar phenomenon has been previously observed for Saccharomyces cerevisiae). Dissolved oxygen levels should be kept constant and high (DO > 40%). To this end, for this particular cell line, it is recommended to increase the gas flow rate from 0.05 vvm to 0.1 vvm (6 L / h for a 1 L working volume) and / or to use only pure oxygen compressed gas instead of compressed air as the oxygen supply gas. This should ensure high oxygen levels throughout the cell culture and prevent lactate production.

[0245] Thus, it was shown that, with respect to the use of the methods and apparatus in connection with mammalian cell culture, utilizing a module to generate PEMF, over three days of the product, an increase in metabolic rate was induced, as seen by accelerated cell growth, which increased by 27% on day 3, while maintaining IgG concentration and production, which is a significant benefit, as all cells and conditions were identical except for the use of PEMF, thereby demonstrating that the use of the methods and apparatus according to the present invention stimulated a higher metabolic activity, as shown by an increase in expression levels per cell, and increased cellular metabolism by approximately 30% through a significant acceleration of cell division, and demonstrated a significant increase of approximately 25% in cellular expression of IgG, resulting in an overall potential 60+% productivity increase (30% more cells, 25% more production per cell).

Claims

1. An apparatus for allowing pulsed electromagnetic waves to be applied to a cell culture for a period of time to change the state of the cell culture, the apparatus comprising at least one support and a container in which the cell culture is located, and wherein, The support comprises one or more modules for generating pulsed electromagnetic waves, and the support comprises or is connected to a control device to control the generation of pulsed electromagnetic waves, and is positionable relative to the cell culture to allow the cell culture to be exposed to the generated pulsed electromagnetic waves, and wherein the one or more modules comprise an antenna and a transmitter for transmitting the pulsed electromagnetic waves within a specific frequency range from the one or more modules to the cell culture, wherein changing the state of the cell culture is changing the metabolic productivity and / or productivity of the cell culture.

2. The device according to claim 1, characterized in that The control device is configured to control the frequency and pulse sequence of the pulsed electromagnetic waves.

3. The device according to claim 1, characterized in that The control device includes a software-based user interface configured to allow a user to control the generation of the pulsed electromagnetic waves through the software-based user interface.

4. The device according to claim 1, characterized in that A plurality of said modules are arranged on said support in a fixed array or configuration to provide increased range and / or intensity of the pulsed electromagnetic waves.

5. The device according to claim 1, characterized in that The container has walls, and the at least one support is located outside the container, and the pulsed electromagnetic waves are applied to the cell culture through one or more walls of the container in which the cell culture is located.

6. The device according to claim 1, characterized in that At least a portion of the at least one support including one or more modules for generating the pulsed electromagnetic waves is located inside the container.

7. The device according to claim 6, characterized in that The at least one support comprises a plurality of supports, each support comprising the one or more modules, wherein each support of the plurality of supports is located at a different position relative to the container.

8. The device according to claim 1, characterized in that The at least one support is formed by one or more walls of the container, and the module is mounted as part of the one or more walls.

9. The device according to claim 1, characterized in that The at least one support is located within one or more walls of the container.

10. The device according to claim 1, characterized in that The at least one support is provided in the form of a housing in which the one or more modules are located.

11. The device according to claim 1, characterized in that The support is provided as a sheet on which the modules lie.

12. The device according to claim 1, characterized in that The support is provided for use in a sterile form and is provided for single use.

13. The device according to claim 1, characterized in that The specific frequency range is the Industrial, Scientific and Medical (ISM) short-range radio band.

14. The device according to claim 1, characterized in that The specific frequency is 2.4 GHz.

15. The device according to claim 1, characterized in that The transmitter of the pulsed electromagnetic waves is capable of generating a pulsed electromagnetic field (PEMF) at a distance of up to 15 meters.

16. The device according to claim 1, characterized in that The control device allows the pulsed electromagnetic wave to be transmitted in pulses, the duration of the pulses being in the range of 0.5-1.5 ms.

17. The device according to claim 16, characterized in that The pulses were separated by rest periods in the range of 40-66 ms.

18. The device according to claim 1, characterized in that The pulses of the pulsed electromagnetic waves are emitted in the range of 12-20 pulses per second.

19. The device according to claim 1, characterized in that The at least one support and / or one or more modules located thereon are arranged relative to the container so as to generate pulsed electromagnetic waves for applying a pulsed electromagnetic field (PEMF) to the cell culture in an omnidirectional manner.

20. A method for changing the state of a cell culture using the device according to any one of claims 1 to 19, wherein The method comprises the steps of applying a pulsed electromagnetic field (PEMF) to a cell culture when in a first state by transmitting pulsed electromagnetic waves from one or more modules at a predetermined frequency and for a predetermined time period to change the first state of the cell culture to a desired second state for subsequent use or further processing, wherein the one or more modules comprise antennas and transmitters to allow transmission of the pulsed electromagnetic waves from the one or more modules to the cell culture within a specific frequency range, wherein changing the state of the cell culture is changing the metabolic productivity and / or productivity of the cell culture.

21. The method according to claim 20, characterized in that Application of pulsed electromagnetic waves to increase the growth rate of mammalian cell cultures.

22. The method according to claim 20, characterized in that The frequency of the pulsed electromagnetic waves is within the bandwidth of the electromagnetic spectrum used for industrial scientific and medical purposes.

23. The method according to claim 20, characterized in that The pulsed electromagnetic waves can be delivered in pulses having a duration in the range of 0.5-1.5 ms.

24. The method according to claim 23, wherein The pulses were separated by rest periods in the range of 40-66 ms.

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