System and method for low-cost sterilization of liquids

Abstract

Provided are methods and systems for low-cost sterilization of media using passive diffusion of media components across a porous membrane.

Description

Attorney Docket No.121430-791899 SYSTEM AND METHOD FOR LOW-COST STERILIZATION OF LIQUIDS FIELD

[0001] The present invention relates to methods and systems for sterilization of non-sterile medium using diffusion through a porous membrane. BACKGROUND

[0002] Currently, factory farming is the primary way most animal products are produced around the world, and, in particular, in the United States of America. However, factory farming is rife with ethical and environmental issues, especially since global demands have increased dramatically. In the recent years, the meat industry particularly has been moving away from factory farming and towards the preparation of protein products that mimic animal-based products, for instance plant-based protein. However, it is notably difficult for such alternative or substitute protein products to achieve the same texture, flavor, and nutritional benefits as protein derived from animals.

[0003] Cultured meat, also known as cell-based, lab-grown, or cultivated meat, is meat that is produced through cellular agriculture techniques using in vitro cell culture or bioreactor systems, rather than being harvested from live animals. Cultured meat produced from cell cultures provides a viable alternative to meat prepared through typical factory farming methods without having to turn to a completely vegetarian or vegan diet. Assessments have shown that cultured meat has the capacity to significantly reduce land and water use, emit fewer greenhouse gases, and reduce agriculture-related pollution and eutrophication. It also provides a more ethical way of producing food, avoiding animal confinement, mistreatment of animals and animal slaughter. In particular, cultured meat can be obtained from animal-based components, but with the advantage of sustainably increased yields, similarity of underlying structure and nutrition to factory farmed animals. In addition, cultured animal cells can also be used for production of other animal-based products like leather, horns, or feathers. Industrialized animal cell culture has also proven to be useful for medical purposes, for example for production of recombinant proteins, tissues, organs and organoids.

[0004] However, for cellular agriculture to be a viable alternative, operation costs must remain low despite large production volumes. This requires development of new methods at each step of the production process allowing for easy scale up without increasing costs. One step that is associated with prohibitively high costs is media sterilization and processing. Thus far, the 1 95815355.1Attorney Docket No.121430-791899 industry has relied on conventional filtration methods for media sterilization. However, these methods, which rely on use of pressure pumps to pass large volumes of media through disposable membranes which quickly clog and require frequent replacement, are not particularly suited for large scale production of commodity products such as meat. As a result, the current methods for large scale sterilization of media are time consuming, costly, and wasteful.

[0005] Therefore, there is a need in the field for developing new methods for large scale sterilization of media. SUMMARY OF THE INVENTION

[0006] In some aspects, the current disclosure encompasses a method for sterilizing an non- sterile medium, the method comprising diffusing the non-sterile medium through a porous membrane, wherein the porous membrane has a pore size of less than 450 nm. In some aspects, the method for sterilizing an non-sterile medium as provided herein comprises the steps of: (a) providing a device configured for sterilizing an non-sterile media, the device comprising a first chamber configured to receive the non-sterile medium, a second chamber comprising a fluid, wherein the second chamber is in fluid communication with the first chamber via a porous membrane, and the porous membrane configured to provide a diffusion window between the first chamber and the second chamber, wherein the porous membrane has a pore size of less than about 450 nm, (b) supplying the non-sterile medium into the first chamber; and (c) diffusing the non-sterile medium through the porous membrane and into the second chamber, wherein a sterilized medium is formed in the second chamber. In some aspects, the fluid in the second chamber is sterile. In one aspect, the fluid in the second chamber is configured to receive a molecule, an ion, a particle, or any combination thereof from the non-sterile medium through the porous membrane, and wherein the fluid in the second chamber has a lower concentration of the molecule, the ion, the particle, or any combination thereof as compared to the non-sterile medium. In some aspects, diffusing the non-sterile medium through the porous membrane into the second chamber is accomplished without the use of a pump.

[0007] In some aspects, the porous membrane has a pore size of less than about 210 nm. Depending on the media and the size of molecules to be retained or allowed to diffuse, membranes with appropriate molecular weight cut-off (MWCO) may be selected as provided herein. 2 95815355.1Attorney Docket No.121430-791899

[0008] Thus, in some aspects, the membrane has a molecular weight cut-off (MWCO) of less than 500 kDa. In some aspects, the membrane is comprised of natural and synthetic proteins such as cellulose, polyvinylidene difluoride (PVDF), fluoropolymer, cellular acetate, composite, polyethersulfone (PES), polyacrylonitrile or a combination thereof. In some aspects, the membrane is a tubular, cylindrical, capillary, spiral wound, single leaf, plate, composite single layered (CSL), or a hollow fiber membrane.

[0009] In some aspects, any non-sterile medium can be sterilized using this method, using an appropriate membranes and suitable sterile liquid in the second chamber to enhance diffusion across the membrane. In some aspects of the method as disclosed herein, the non-sterile medium is a heat-sensitive medium. In some aspects, the non-sterile medium is a radiation sensitive or chemical treatment sensitive medium. In some aspects, the non-sterile medium is a basal medium, spent medium, composite medium, or conditioned medium. In some aspects, basal medium is selected from DMEM, DMEM F12, minimum essential medium, RMI 1640, serum free medium, bench stable medium, human plasma like medium, primary culture medium, or supplemented culture medium. In some aspects, the non-sterile medium comprises at least one egg protein, or a derivative, variant of fragment thereof. In some aspects, the at least one egg protein is selected from ovalbumin, ovotransferrin, ovomucoid, ovomucin or lysozyme, apovitellenin I-VI, a lipovitellin, a livetin, phosvitin, biotin binding protein or a variant or derivative thereof, or any combination thereof. In some aspects, the non-sterile medium further comprises additional egg-based ingredients. In some aspects, the non-sterile medium further comprises growth factors, amino acids, vitamins, minerals, edible surfactants, antibiotics, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, alginates, collagen, gelatin, poly(amino acids), peptides, polypeptides, fats, fibers, buffering agents or any combinations thereof. In some aspects, the non-sterile medium is spent medium from a bioreactor. In some aspects, the non-sterile medium contains nanoparticles made of metals, fat, protein, emulsified oils.

[0010] In some aspects of the method as disclosed herein, the first chamber is in fluid communication with a feed tank through an inlet port and the second chamber is in fluid communication with a bioreactor through an outlet port, such that the method is operated essentially as a closed loop. In some aspects, the method further comprises the step of clearing the membrane of particulate matter and debris with a circulating flow system. In some aspects, the method does not require a heating system for sterilizing the non-sterile medium. In some aspects, the method does not require chemical treatment of the medium. In some aspects, the 3 95815355.1Attorney Docket No.121430-791899 method does not require radiation treatment of the medium. In some aspects, the method as disclosed herein, reduces cost of sterilization of the medium by at least 20%-99%.

[0011] In some aspects, the method further comprises the step of: circulating the non-sterile medium through the first chamber, wherein the first chamber comprises a manifold of pipes made of porous membrane, and wherein the manifold of pipes is in fluid communication with the second chamber. In some aspects, the method further comprises the step of: circulating the non-sterile medium through the first chamber, wherein the first chamber comprises a manifold of pipes comprising a porous membrane, the manifold of pipes forming the second chamber, and wherein the first chamber is in fluid communication with the manifold of pipes through the porous membrane. In some aspects, the method further comprises adding the molecule, the ion, the particle, or a combination thereof to the non-sterile medium to facilitate diffusion from the first chamber into the second chamber through the porous membrane.

[0012] In some aspects, the current disclosure also encompasses a system for sterilizing an non-sterile media, the system comprising: (a) a device configured for sterilization of an non-sterile media, the device comprising: (b) a first chamber configured to receive the non-sterile medium; (c) a second chamber containing a fluid; and a porous membrane separating the first chamber from the second chamber and configured to provide a diffusion window between the first chamber and the second chamber, wherein the first chamber is in fluid communication with the second chamber via the porous membrane, and the porous membrane has a pore size of less than 450 nm. In some aspects, the system further comprises an inlet port configured to be fluidly connected to the first chamber for supplying the non-sterile medium into the first chamber. In some aspects, the system further comprises an outlet port configured to be fluidly connected to the second chamber for removing the fluid from the second chamber. In some aspects of the system as disclosed herein, the concentration of at least a molecule, an ion, a particle, or any combination thereof in the fluid is less than or equal to the concentration of the molecule, the ion, the particle, or any combination thereof in the non-sterile medium. In some aspects, the first chamber comprises a manifold containing at least one pipe, wherein the manifold is in fluid communication with the second chamber via the porous membrane. In some aspects, the system as disclosed herein further comprises a circulating flow system to remove particulate matter and debris from a surface of the porous membrane. In some aspects, the inlet port or outlet port or both are in fluid communication with a bioreactor. In some aspects, the system is essentially a closed system. In some aspects, the system is an open system. 4 95815355.1Attorney Docket No.121430-791899

[0013] In some aspects of the system as disclosed herein, the porous membrane has a pore size of less than about 210 nm. In some aspects, the porous membrane has a molecular weight cut-off (MWCO) of less than 500 kDa. In some aspects, the membrane is comprised of polyvinylidene difluoride (PVDF), fluoropolymer, cellular acetate, composite, polyethersulfone (PES), Polyacrylonitrile or a combination thereof. In some aspects, the membrane is a tubular, cylindrical, capillary, spiral wound, single leaf, plate, layered, or a hollow fiber membrane or a combination thereof.

[0014] In some aspects, the system as disclosed herein further comprises a waste port configured to remove solid waste from the system. In some aspects, the non-sterile medium is sensitive to one or more of heat, radiation or specific chemicals used in media sterilization. In some aspects, the non-sterile medium is a basal medium, spent medium, composite medium, or conditioned medium. In some aspects, the basal medium is selected from DMEM, DMEM F12, minimum essential medium, RMI 1640, serum free medium, bench stable medium, human plasma like medium, primary culture medium, or supplemented culture medium. In some aspects, the non-sterile medium comprises at least one egg protein, or a derivative, variant of fragment thereof. In some aspects, the at least one egg protein is selected from ovalbumin, ovotransferrin, ovomucoid, ovomucin or lysozyme, apovitellenin I-VI, a lipovitellin, a livetin, phosvitin, biotin binding protein or a variant or derivative thereof, or any combination thereof. In some aspects, the non-sterile medium further comprises additional egg-based ingredients. In some aspects, the non-sterile medium further comprises growth factors, amino acids, vitamins, minerals, edible surfactants, antibiotics, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, alginates, collagen, gelatin, poly(amino acids), peptides, polypeptides, fats, fibers, buffering agents or any combinations thereof.

[0015] In some aspects of the system as disclosed herein, the system does not use a pressure pump for passing the non-sterile medium through the porous membrane. In some aspects, the system does not require one or more of heat, radiation, or chemical treatment for sterilizing non- sterile media. In some aspects, the system is for use in cellular agriculture applications.

[0016] In some aspects, the current disclosure also encompasses methods for cellular agriculture, the method comprising, using the systems as disclosed herein to obtain a sterilized medium. In some aspects, the method reduces cost of sterilization of the non-sterile medium by at least about 20%-99%. 5 95815355.1Attorney Docket No.121430-791899 BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A provides a schematic representation of a sterilization method wherein non- sterile fetal bovine serum (FBS) is placed in a first chamber, in fluid communication with a second chamber comprising sterile water through a porous membrane. With the passing of time, components of the non-sterile FBS pass through the membrane achieving a 20% FBS solution in the sterile chamber, that can be used in a media composition.

[0018] FIG. 1B provides a schematic representation of a sterilization method wherein non- sterile fetal basal medium at 4X concentration is placed in a first chamber, in fluid communication with a second chamber comprising sterile water through a porous membrane. With the passing of time, components of the non-sterile basal medium pass through the membrane achieving a 2X basal medium solution in the sterile chamber, that can be used in a media composition.

[0019] FIG.1C provides a schematic representation of mixing of the sterile components of the FBS as provided in FIG.1A and the 2X basal medium as provided in FIG.1B to make a FBS 10% sterile media.

[0020] FIG.2A provides an embodiment of the system as disclosed herein, wherein the system 10 comprises a first chamber 100 configured to receive non-sterile liquid 140 through an inlet port 160, and a second chamber 110 forming a manifold 180 with pipes, the second chamber comprising sterile media or sterile water 130 or combination thereof. The first and the second chamber are in fluid communication through a porous membrane 120. Sterile liquid can be transferred back to the reactor or a receiving unit via an outlet port 170. One or more additional inlet ports, for example 190 may be added to the system for circulation or cleaning purposes.

[0021] FIG.2B provides an embodiment of the system 20 as disclosed herein comprising a first chamber 200 comprising a manifold 270 with pipes configured to receive non-sterile 240 media through an inlet port 250, and a second chamber 210 comprising a sterile liquid 220. The first and second chamber are in fluid communication via the porous membrane 230. Sterile media can be transferred out of the sterilization unit through the outlet port 260. One or more additional inlet ports, for example 280 may be added to the system for circulation or cleaning purposes.

[0022] FIG. 2C provides an embodiment of the closed system as disclosed herein, with a bioreactor 300 and a sterilization unit 310, connected by an inlet port 320 and an outlet port 340. In some aspects, an outlet port to remove solids 330 and additional inlet port 350 may be added. 6 95815355.1Attorney Docket No.121430-791899

[0023] FIG.3A is a schematic representation of the first and second chamber, with the first chamber or the source chamber comprising BSA and polystyrene beads. After 6 hrs the BSA concentration equilibrated between the two chambers.

[0024] FIG.3B is a graphical representation of the diffusion process over time.

[0025] The drawing figures do not limit the present inventive concept to the specific aspects disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain aspects of the present inventive concept. DETAILED DESCRIPTION

[0026] The following detailed description illustrates various aspects of the present inventive concept. The description is intended to describe aspects of the present inventive concept in sufficient detail to enable those skilled in the art to practice the present inventive concept. Other components can be utilized, and changes can be made without departing from the scope of the present inventive concept. The following description is, therefore, not to be taken in a limiting sense. The scope of the present inventive concept is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0027] The present disclosure is based, in part, on the discovery that diffusion-based macromolecule separation using a nanopore membrane can be used to obtain a sterile solution while leaving behind large microorganisms and particles. Experimental determination of speed and efficiency of sterilizing large volumes of media were conducted and are ongoing. These experiments as illustrated in the Examples and the contemplated systems detailed herein provide a cost-effective and low-waste alternative to contemporary filtration methods. I. Methods

[0028] In some aspects, the current disclosure encompasses a method for sterilizing an non- sterile medium, the method comprising diffusing the non-sterile medium through a porous membrane, wherein the porous membrane has a pore size of less than 210 nm. In some aspects, the method encompasses the process of passive diffusion of at least one media component across a membrane, in response to a concentration gradient. Without being bound by theory, diffusion of particles in solution can be analyzed using the kinetic theory of gases, and the general form is: ^^ = μ ^^^^T 7 95815355.1Attorney Docket No.121430-791899

[0029] where D, μ, kB, and T are the diffusion coefficient, the ratio of the particle’s drift velocity to an applied force, Boltzmann’s constant, and absolute temperature, respectively. The Equation is modified to describe the diffusing spherical particles in a liquid, and it is the Stokes–Einstein equation that is widely used: D=k ^^T6πηr

[0030] where η and r are the dynamicradius of the spherical particle, respectively. The diffusion coefficient is a function of the r−1, resulting in the diffusion-based separation that is efficient for small particles. In this study, a separation and sterilization method was developed based on the dominant property of diffusion over sedimentation in nano-sized particles.

[0031] In some aspects, the method as disclosed herein encompasses using at least a two- chamber system, wherein the two chambers are separated by a porous membrane and the first chamber comprises an non-sterile medium, while the second chamber is sterile and comprises a medium which differs from the medium in the first chamber with regards to concentration of at least one component. Exemplary embodiments of the system that may be used to conduct the method disclosed herein are provided.

[0032] In some aspects, the second chamber comprises a medium having a lower concentration of the at least one component as compared to the medium contained in the first chamber. In some aspects, the second chamber comprises water or a fluid that comprises components at a lower concentration than the fluid in the first chamber. In some exemplary set up and purely to illustrate an embodiment of the current disclosure, the method may comprise a set-up as provided in FIG.1.

[0033] In some aspects, the media component that is received by the fluid in the second chamber is a molecule, an ion, a particle, or any combination thereof. Examples of molecules include but are not limited to macromolecules, organic compounds, inorganic compounds, salts, nucleotides, polynucleotides, polypeptides, sugars, buffering components, amino acids, growth factors, vitamins, minerals, fats, edible surfactants, antibiotics, fibers, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, alginates, collagen, gelatin, poly(amino acids), peptides, poly(α-hydroxyacids), polylactic or polyglycolic acids, poly-lactide poly-glycolide copolymers, poly-lactide polyethylene glycol (PEG) copolymers, polyesters, poly(ε- caprolactone), poly(3-hydroxy-butyrate), poly(s-caproic acid), poly(p-dioxanone), poly(propylene fumarate), poly(ortho esters), polyol / diketene acetal addition polymers, polyanhydrides, 8 95815355.1Attorney Docket No.121430-791899 poly(sebacic anhydride) (PSA), poly(carboxybiscarboxyphenoxyphenoxyhexane) (PCPP), poly[bis(p-carboxyphenoxy)methane] (PCPM), copolymers of SA, CPP and CPM poly (amino acids), poly(pseudo amino acids), polyphosphazenes, derivatives of poly[(dichloro)phosphazene], poly[(organo)phosph-azenes]polymers, polyphosphates, polyethylene glycol polypropylene block co-polymers, co-polymers prepared from the monomers of these polymers, random blends of these polymers, or mixtures or combinations thereof. and polysaccharides. In some aspects, the polypeptide may be an egg-based polypeptide. Non- limiting examples of egg-based polypeptides include ovalbumin, ovotransferrin, ovomucoid, ovomucin or lysozyme, apovitellenin I-VI, a lipovitellin, a livetin, phosvitin, biotin binding protein or a variant or derivative thereof, or any combination thereof. In some aspects, the molecule may be a growth factor. Non-limiting examples of growth factors include is FGF, VEGF, PGF, TGF, BDNF, GDNF, Shh, BMP-2, BMP-4, EGF, Noggin, R-Spondin 1, Wnt-3a, FGF-10, FGF-2,FGF-7, Activin A, IGF1, NRG1, Jagged 1, HGF, IL-2, IL-4, IL-6, IL-3, IL-7, Flt3-ligand, PDGF, or TGF-β1 or any combination thereof. Non-limiting examples of vitamins that can be incorporated into the disclosed compositions include vitamin D, A, or different vitamin B complexes, thiamine, riboflavin, folic acid, D-Ca pantothenate. Non-limiting examples of ions include sodium, potassium, ferrous, ferric, manganese, magnesium, zinc, copper, calcium, chloride, sulphates, citrate, bicarbonates, nitrates, or oxalates. Non limiting examples of particles may include polymers, beads, micro / nano carriers, emulsifiers, surfactants, dispersants, scaffolds, etc. In some aspects, the micro / nanocarriers may comprise at least one egg-based protein. In some aspects, the fluid in the second chamber has a lower concentration of said molecule, ion or particle, or a combination thereof than the fluid of the first chamber.

[0034] In an exemplary aspect, the non-sterile media may be any of the media or a media component commonly used in the art. In some aspects, the media or media components may be commercially available. Non-limiting exemplary cell culture media that may be purchased from commercial vendors (e.g., Gibco, Sartorius, etc.), or synthesized, include SAFC Excell media, BME (basal Eagle Medium), MEM (minimum Eagle Medium), medium 199, DMEM (Dulbecco's modified Eagle Medium), GMEM (Glasgow modified Eagle medium), DMEM-HamF12, Ham-F12 (Gibco) and Ham-F10 (Gibco), IMDM (Iscove's Modified Dulbecco's medium), MacCoy's 5A medium, RPMI 1640, RMI 1640, serum free medium, fetal bovine serum (FBS), basal media, bench stable medium, primary culture medium, supplemented culture medium or GTM3 or combinations thereof. In some aspects, the cell culture medium may include additional components including amino acids, proteins, fats, vitamins, minerals, edible surfactants, antibiotics, buffering agents, platelet rich plasma (PRP), platelet poor plasma (referred to as 9 95815355.1Attorney Docket No.121430-791899 plasma), a platelet concentrate, a lysate of red blood cells, a platelet lysate (PL), cytokines, fibers, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, sugars, alginates, collagen, gelatin, polymers or any combination thereof as disclosed herein. In some aspects, the media or media component is not conducive to heat or radiation sterilization.

[0035] Thus, in some aspects, the method as disclosed herein encompasses a method for sterilizing an non-sterile medium, the method comprising providing a device configured for sterilizing an non-sterile media, the device comprising a first chamber configured to receive the non-sterile medium, a second chamber comprising a fluid, wherein the second chamber is in fluid communication with the first chamber via a porous membrane, and the porous membrane configured to provide a diffusion window between the first chamber and the second chamber, wherein the porous membrane has a pore size of less than about 210 nm; supplying the non- sterile medium into the first chamber; and diffusing the non-sterile medium through the porous membrane and into the second chamber, wherein a sterilized medium is formed in the second chamber. In some aspects of the method disclosed herein, the diffusion is passive diffusion and does not require the use of a pressure pump to transport the media across the membrane.

[0036] In some aspects, the porous membrane that allows fluid communication between the first and the second chamber has a pore size of less than 210 nm. In some aspects, the pore size is configured to not enable any living microorganism, for example bacteria, virus, bacteriophage, algae or protozoa, to pass through the membrane. In some aspects, the membrane has a pore size of between 50 nm to about 200 nm. In some aspects, the membrane has a pore size of about 50 nm to about 70 nm, or about 70 nm to about 90 nm, or about 90 nm to about 110 nm, or about 110 nm to about 130 nm, or about 130 nm to about 150 nm, or about 150 nm to about 170 nm, or about 170 nm to about 200 nm, or about 200 nm to about 230 nm.

[0037] Depending on the media and the size of molecules to be retained or allowed to diffuse, membranes with appropriate molecular weight cut-off (MWCO) may be selected (Erickson 2009, Biol Proced Online; 11:32-51). For example, in cases of proteins, the following formulae can be used to determine the appropriate MWCO, where V is the volume of the protein molecule and M is the MW:

[0038] Assuming the a a minimal radius can be Rminmay be calculated: 10 95815355.1Attorney Docket No.121430-791899

[0039] Thus, in some aspects, the hasweight cut-off (MWCO) less than 500 kDa. In some aspects, the membrane has a molecular weight cut-off of about 5 kDa to about 500 kDa. In some aspects, the membrane has a molecular weight cut-off of about 10 kDa to about 100 kDa. In some aspects, the membrane has a molecular weight cut-off of about 5kDa to about 10 kDa, of about 10 kDa to about 50 kDa, or 50 kDa to about 100 kDa, or about 100 kDa to about 150 kDa, or about 150 kDa to about 200 kDa, or about 200 kDa to about 250 kDa, or about 250 kDa to about 300 kDa, or about 300 kDa to about 350 kDa, or about 350 kDa to about 400 kDa, or about 400 kDa to about 450 kDa, or about 450 kDa to about 500 kDa.

[0040] In some aspects, the membrane may be a commercially available membrane. In some aspects the membrane may be, for example, a polyvinylidene difluoride (PVDF), a fluoropolymer, a cellular acetate, a composite, a polyethersulfone (PES), a polyacrylonitrile membrane or a combination thereof. In some aspects, the membrane may be, for example, configured in a tubular, cylindrical, capillary, spiral wound, single leaf, plate, layered, or a hollow fiber membrane format or a combination thereof. In some aspects, the configuration of the membrane is such that it maximizes the contact area between the fluid and the membrane. In some aspects, the porous membrane may be placed as a continuous membrane between the two chambers or the membrane may be a discontinuous membrane.

[0041] In some aspects, the method may further comprise filtering the medium through a mesh to remove particulate matter prior to diffusion of the filtered medium through the porous membrane. In some aspects, the method further comprises clearing the membrane of debris with a circulating flow system. In some aspects, the circulating flow system is a passive flow system. In some aspects, the circulating flow system is a continuous flow system. In some aspects, the circulating flow system is a discontinuous flow system and used between operations. In some aspects, the flow system may utilize an active pump for clearing the membrane between operations.

[0042] In some aspects, the solid or concentrate solute may be added to source (non-sterile compartment) to compensate for dilution through the diffusion of water in a batch mode or a continuous flow manner. In some aspects, the non-sterile liquid such as serum are constantly concentrated by vacuum evaporation system or similar low temperature technologies to compensate for dilution through the diffusion of water in a batch mode or continuous flow manner. 11 95815355.1Attorney Docket No.121430-791899

[0043] In some aspects, the method for sterilization of media as disclosed herein is useful for cellular agriculture. In some aspects, the sterilized medium is for use in a bioreactor. In some aspects, the sterilization system as disclosed herein may operate as a continuous flow system, operating in a closed loop with a bioreactor. Thus, in some aspects of the current method, the first chamber is in fluid communication with a feed tank through an inlet port and the second chamber is in fluid communication with a bioreactor through an outlet port, such that the method is operated essentially as a closed loop. In some aspects, the feed tank provides spent media from the bioreactor after the removal of cell product. The spent media is in fluid communication with the sterile media via a porous membrane, and components from the spent media can passively diffuse across the membrane thus allowing re-use of spent media and reduction in processing costs. In some aspects, the method operates as an open loop, wherein the sterilized media is harvested and used when necessary.

[0044] In some aspects, the current disclosure also encompasses media products that have been sterilized using the methods disclosed herein.

[0045] In some aspects, the method disclosed herein significantly reduces operation costs for cellular agriculture. Current methods used rely on active pumps that tend to clog membranes, thereby requiring the process to stop while the membrane is unclogged or replaced. Additionally, the cost of maintenance is higher in an active pump system. Alternatively, passive diffusion methods provide a cost-effective alternative and can help reduce costs by about 20% to about 99%. In some aspects, the cost reduction achieved can be between 20% to 30%, or 30% to 40%, or 40% to 50%, or 50% to 60%, or 60% to 70%, or 70% to 80%, or 80% to 90%, or 90% to 99%. In some aspects, the current disclosure also encompasses a method of sterilization media, wherein the method reduces cost of the media by about 20% to 30%, or 30% to 40%, or 40% to 50%, or 50% to 60%, or 60% to 70%, or 70% to 80%, or 80% to 90%, or 90% to 99%.

[0046] Some exemplary use cases for the method disclosed herein are provided in FIG.1A- 1C. As seen in FIG.1A, non-sterile FBS is placed in a first chamber, and sterile water is placed in a second chamber which is adjacent to the first chamber thereby allowing diffusion of components from FBS to the sterile water through a porous membrane. Over time, the components of FBS accumulate in the sterile water contained in the second chamber, resulting in a 20% FBS solution (see FIG. 1A). In another exemplary use case, concentrated 4X basal medium is sterilized by diffusion through a membrane to obtain 2X diluted ready-to-use sterile medium (FIG. 2B). The two components can then be mixed to obtain a culture medium as provided in FIG.1C. 12 95815355.1Attorney Docket No.121430-791899 II. Systems

[0047] In some aspects, the current disclosure encompasses systems to perform the methods as disclosed herein. In some aspects the current disclosure encompasses a system for sterilizing an non-sterile media, the system comprising: a device configured for sterilizing an non-sterile media, the device comprising: a first chamber configured to receive the non-sterile medium; a second chamber containing a fluid; and a porous membrane separating the first chamber from the second chamber and configured to provide a diffusion window between the first chamber and the second chamber, wherein the first chamber is in fluid communication with the second chamber via the porous membrane, and the porous membrane has a pore size of less than 450 nm and preferably less than 210 nm. In one aspect, the sterilization system as disclosed herein does not comprise a pressure pump.

[0048] In one aspect, the system may further comprise an inlet port configured to receive non- sterile or non-sterile medium and connected to a first chamber for transferring the non-sterile medium into the first chamber. In some aspects, the system may further comprise an outlet port configured to receive sterilized medium transferred from the second chamber and out of the sterilization system.

[0049] In some aspects, the first chamber may comprise a manifold containing at least one pipe, wherein the manifold is placed in fluid communication with the second chamber via the porous membrane. Without being bound by theory, the splitting of the manifold into pipes provides a larger surface area for placing the membrane and improves diffusion rates. In some aspects, the second chamber may comprise a manifold split into at least one pipe.

[0050] In some aspects of the system, the inlet port or the outlet port or both the inlet port and outlet port may comprise a valve for regulating the flow of the medium into and out of the system.

[0051] In some aspects, the sterilization system as disclosed herein may be connected to a bioreactor. In some aspects, the inlet port of the sterilization system may be configured to receive spent media from the bioreactor following the removal of cellular biomass. Thus, in some aspects, the system as disclosed herein may further comprise ports to remove solids / biomass from the system. In some aspects, the outlet port of the sterilization system may be configured to remove sterilized media from the sterilization system into the bioreactor. In some aspects, the system comprising the bioreactor and the sterilization system may function as an essentially closed system but having at least one additional port configured to remove cellular biomass from the system. In one aspect, the system may be an open system. 13 95815355.1Attorney Docket No.121430-791899

[0052] In some aspects, the system operates like a flow system circulating media from the reactor, into the sterilization unit, and back into the bioreactor. In some aspects, the system may further comprise a circulating system for removing debris or particulate matter or both from the surface of the porous membrane. In one aspect, the circulating system for removal of debris or particulate matter or both from the surface of the porous membrane may be activated between operations.

[0053] Some exemplary system set-ups are provided in FIG. 2A-2C. As provided in the exemplary configuration depicted in FIG. 2A, in one aspect, the system 10 may comprise a lower / outer chamber 100 configured to receive a non-sterile liquid 140 through an inlet port 160, and an upper / inner chamber 110 that may be split into manifolds 180 to increase surface area, the inner chamber comprising a sterile fluid 130. The inner and outer chambers 100, 110 are in fluid communication through a 210 nm membrane 120. Sterilized medium can then be transferred back into a reactor or a receiving unit via an outlet port 170. In some aspects, the system may comprise additional inlet port 190 to facilitate closed loop circulation of media or cleaning.

[0054] In another exemplary set-up which is depicted in FIG.2B the system 20 may comprise a lower / inner chamber 200 split into manifolds 270 configured to receive non-sterile media 240 through an inlet port 250, and an upper / outer chamber 210 comprising a sterile liquid 220. The outer and inner chambers 210, 200 are in fluid communication via a porous membrane 230. Sterilized media can be transferred out of the sterilization system through the outlet port 260. In some aspects, the system may comprise additional inlet port 280 to facilitate closed loop circulation of media or cleaning.

[0055] In another exemplary set-up as depicted in FIG.2C, the system 30 operates as a closed loop system, wherein the system comprises a reactor 300 and a sterilization chamber 310 connected through an inlet port 320 and an outlet port 340. In some aspects, an outlet port to remove solids 330 and additional inlet port 350 may be added. These disclosed systems are exemplary embodiments and should not be considered limiting.

[0056] In some aspects of the system as disclosed herein, the first and second chambers are in fluid communication via a porous membrane having a pore size configured to not enable viruses, bacteria, fungal spores, algae or protozoa to pass through. In some aspects, the membrane has a pore size of less than 450 nm. In some aspects, the membrane has a pore size of between 50 nm to about 450 nm in size. In some aspects, the membrane has a pore size of about 50 nm to about 70 nm, or about 70 nm to about 90 nm, or about 90 nm to about 110 nm, or about 110 nm to about 130 nm, or about 130 nm to about 150 nm, or about 150 nm to about 14 95815355.1Attorney Docket No.121430-791899 170 nm, or about 170 nm to about 200 nm, or about 200 nm to about 230 nm, or about 230 nm to about 260 nm, or about 260 nm to about 290 nm, or about 290 nm to about 310 nm, or about 310 nm to about 340 nm, or about 340 nm to about 370 nm, or about 370 nm to about 400 nm, or about 400 nm to about 450 nm. In some aspects, the membrane has a pore size of less than 210 nm.

[0057] In some aspects, the membrane has a molecular weight cut-off of about 5 kDa to about 500 kDa. In some aspects, the membrane has a molecular weight cut-off of about 5kDa to about 10 kDa, of about 10 kDa to about 50 kDa, or 50 kDa to about 100 kDa, or about 100 kDa to about 150 kDa, or about 150 kDa to about 200 kDa, or about 200 kDa to about 250 kDa, or about 250 kDa to about 300 kDa, or about 300 kDa to about 350 kDa, or about 350 kDa to about 400 kDa, or about 400 kDa to about 450 kDa, or about 450 kDa to about 500 kDa..

[0058] In some aspects, the membrane may be made from a natural material, non-limiting example of which include cellulose. In some aspects, the membrane may be made from a synthetic material, for example polyvinylidene difluoride. In some aspects, the membrane may be made from a semi-synthetic material, for example cellulose acetate. In some aspects, the membrane may be a commercially available membrane. In some aspects, the membrane may be, for example, a polyvinylidene difluoride (PVDF), a fluoropolymer, a cellular acetate, a composite, a polyethersulfone (PES), a polyacrylonitrile membrane or a combination thereof. In some aspects, the membrane may be, for example, configured in a tubular, cylindrical, capillary, spiral wound, single leaf, plate, layered, or a hollow fiber membrane format or a combination thereof. In some aspects, the configuration of the membrane is such that it maximizes the contact area between the fluid and the membrane. In some aspects, the porous membrane may be placed as a continuous membrane between the two chambers or may be a discontinuous membrane.

[0059] In one aspect, the system as disclosed herein is configured to carry out the method as disclosed in section I of the detailed description. In some aspects, the system comprises a first chamber comprising a non-sterile medium, and a second chamber comprising a sterile medium, wherein the concentration of least a molecule, an ion, a particle, or any combination thereof in the sterile medium is less than or equal to the concentration of the molecule, the ion, the particle, or any combination thereof in the non-sterile medium.

[0060] In some aspects, the current disclosure also encompasses use of the systems and methods disclosed herein for the production of sterilized medium for making cell culture meat products. In some aspects, the current disclosure also encompasses the use of the disclosed systems and method or a combination thereof for production of structural animal products. 15 95815355.1Attorney Docket No.121430-791899

[0061] In some aspects, the current disclosure also encompasses the use of the disclosed systems and methods for production of media for culturing of cells, organs, tissues, or organoids for medical purposes. In some aspects, the current disclosure also encompasses use of the media for production of cell products including but not limited to proteins, glycoproteins, polysaccharides, lipids, biomass, or cells. III. Terminology

[0062] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present inventive concept or the appended claims.

[0063] Further, as the present inventive concept is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present inventive concept and not intended to limit the present inventive concept to the specific aspects shown and described. Any one of the features of the present inventive concept may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present inventive concept may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present inventive concept will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present inventive concept, and be encompassed by the claims.

[0064] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact 16 95815355.1Attorney Docket No.121430-791899 configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0065] The terms "comprising," "including" and "having" are used interchangeably in this disclosure. The terms "comprising," "including" and "having" mean to include, but not necessarily be limited to the things so described.

[0066] Lastly, the terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in some way inherently mutually exclusive. EXAMPLES

[0067] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure. EXAMPLE 1: Viability of diffusion through a nanopore membrane for macromolecular separation

[0068] It was contemplated that diffusion-based macromolecule separation using a nanopore membrane would be advantageous to obtain sterile solution while leaving behind large microorganism and particles. To compare the separation speed of diffusion-based separation, the mathematical model for each method was derived. The model for analyzing sedimentation is 17 95815355.1Attorney Docket No.121430-791899 closely related to the particle size, density, and viscosity of the fluids. The equation for spherical particle sedimentation velocity in centrifuge-based separation is given as follows: 6g(ρ 2^^ =p − ρf) ^^η (1) where g, ρp, ρf, r, and η are thefluid, radius of a spherical particle, and dynamic viscosity of the fluid, respectively. The sedimentation velocity is a function of the r2, resulting in separation that is efficient for large (>1 μm) particles. On the other hand, diffusion of particles in solution can be analyzed with the kinetic theory of gases, and the general form is: ^^ = μ ^^^^T (2) where D, μ, kB, and T are the diffusion coefficient, the ratio of the particle’s drift velocity to an applied force, Boltzmann’s constant, and absolute temperature, respectively. The Equation (2) is modified to describe the diffusing spherical particles in a liquid, and it is the Stokes–Einstein equation that is used: D=k ^^T(3) where η and r are the dynamicdiffusion coefficient is a function of the r−1, resulting in the diffusion-based separation that is efficient for small particles. In this study, a separation and sterilization system was developed based on the dominant property of diffusion over sedimentation in nano-sized particles. Bovine serum albumin (BSA) nanosized protein agglomerate was diffused through the separation membrane cartridge having pore size of 200 nm to confirm the separation efficiency. Results show that 6 hours would be enough time for separating BSA protein from the non-sterile BSA sample contaminated with ~1 µm polystyrene beads (FIG.3A). Initial concentration of BSA was set at 40% w / v in source chamber, while the harvest was measured at 20% w / v after 6 hours (FIG.3B). 18 95815355.1

Claims

Attorney Docket No.121430-791899 Claims:

1. A method for sterilizing a non-sterile medium, the method comprising diffusing the non- sterile medium through a porous membrane, wherein the porous membrane has a pore size of less than 450 nm.

2. A method for sterilizing a non-sterile medium, the method comprising: a. providing a device configured for sterilizing a non-sterile media, the device comprising: i. a first chamber configured to receive the non-sterile medium; ii. a second chamber comprising a fluid, wherein the second chamber is in fluid communication with the first chamber via a porous membrane; and iii. the porous membrane configured to provide a diffusion window between the first chamber and the second chamber, wherein the porous membrane has a pore size of less than about 450 nm; b. supplying the non-sterile medium into the first chamber; and c. diffusing the non-sterile medium through the porous membrane and into the second chamber, wherein a sterilized medium is formed in the second chamber.

3. The method of claim 2, wherein the fluid in the second chamber is sterile.

4. The method of claim 2, wherein the fluid in the second chamber is configured to receive a molecule, an ion, a particle, or any combination thereof from the non-sterile medium through the porous membrane, and wherein the fluid in the second chamber has a lower concentration of the molecule, the ion, the particle, or any combination thereof as compared to the non-sterile medium.

5. The method of any one of claims 1-4, wherein diffusing the non-sterile medium through the porous membrane into the second chamber is accomplished without use of a pump pressure across the membrane.

6. The method of any one of claims 1-5, wherein the porous membrane has a pore size in the range of less than about 210 nm.

7. The method of claim 6, wherein the membrane has a molecular weight cut-off (MWCO) is less than 500 kDa. 19 95815355.1Attorney Docket No.121430-791899 8. The method of any of claims 6 or 7, wherein the porous membrane is comprised of natural, synthetic, or semisynthetic polymers.

9. The method of any of claims 6 or 7, wherein the porous membrane is comprised of polyvinylidene difluoride (PVDF), fluoropolymer, cellulose acetate, composite, polyethersulfone (PES), Polyacrylonitrile or a combination thereof.

10. The method of any one of claims 5 or 6, wherein the porous membrane is a tubular, cylindrical, capillary, spiral wound, single leaf, plate, composite single layered (CSL), or a hollow fiber membrane.

11. The method of any one of claims 1 or 2, wherein the non-sterile medium is a heat sensitive medium.

12. The method of claim 10, wherein the non-sterile medium is a basal medium, spent medium, composite medium, or conditioned medium.

13. The method of claim 11, wherein the basal medium is selected from DMEM, DMEM F12, minimum essential medium, RMI 1640, serum free medium, bench stable medium, human plasma like medium, primary culture medium, or supplemented culture medium.

14. The method of claim 10 or claim 11, wherein the non-sterile medium comprises at least one egg protein, or a derivative, variant of fragment thereof.

15. The method of claim 13, wherein the at least one egg protein is selected from ovalbumin, ovotransferrin, ovomucoid, ovomucin or lysozyme, apovitellenin I-VI, a lipovitellin, a livetin, phosvitin, biotin binding protein or a variant or derivative thereof, or any combination thereof.

16. The method of any one of claims 13 or 14, wherein the non-sterile medium further comprises additional egg-based ingredients.

17. The method of any one of claims 10-15, wherein the non-sterile medium further comprises growth factors, amino acids, vitamins, minerals, edible surfactants, antibiotics, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, alginates, collagen, gelatin, poly(amino acids), peptides, polypeptides, fats, fibers, buffering agents or any combinations thereof. 20 95815355.1Attorney Docket No.121430-791899 18. The method of claim 11, wherein the non-sterile medium is spent medium from a bioreactor.

19. The method of claim 2, wherein the first chamber is in fluid communication with a feed tank through an inlet port and the second chamber is in fluid communication with a bioreactor through an outlet port, such that the method is operated essentially as a closed loop.

20. The method of claim 2, wherein the method further comprises clearing the membrane of particulate matter and debris with a circulating flow system.

21. The method of claim 1 or 2, wherein the method does not require a heating system for sterilizing the non-sterile medium.

22. The method of claim 1 or 2, wherein use of the method reduces cost of sterilization of the medium by at least 20%-99%.

23. The method of claim 2, wherein the method further comprises: circulating the non-sterile medium through the first chamber, wherein the first chamber comprises a manifold of pipes made of porous membrane, and wherein the manifold of pipes is in fluid communication with the second chamber.

24. The method of claim 2, wherein the method further comprises: circulating the non-sterile medium through the first chamber, wherein the first chamber comprises a manifold of pipes comprising a porous membrane, the manifold of pipes forming the second chamber, and wherein the first chamber is in fluid communication with the manifold of pipes through the porous membrane.

25. The method of claim 4, wherein the method further comprises adding the molecule, the ion, the particle, or a combination thereof to the non-sterile medium to facilitate diffusion from the first chamber into the second chamber through the porous membrane.

26. A system for sterilizing a non-sterile media, the system comprising: a. a device configured for sterilization of a non-sterile media, the device comprising: i. a first chamber configured to receive the non-sterile medium; ii. a second chamber containing a fluid; and 21 95815355.1Attorney Docket No.121430-791899 iii. a porous membrane separating the first chamber from the second chamber and configured to provide a diffusion window between the first chamber and the second chamber, wherein the first chamber is in fluid communication with the second chamber via the porous membrane, and the porous membrane has a pore size of less than 450 nm.

27. The system of claim 25, further comprising an inlet port configured to be fluidly connected to the first chamber for supplying the non-sterile medium into the first chamber.

28. The system of claim 25, further comprising an outlet port configured to be fluidly connected to the second chamber for removing the fluid from the second chamber.

29. The system of claim 25, wherein concentration of at least a molecule, an ion, a particle, or any combination thereof in the fluid is less than or equal to the concentration of the molecule, the ion, the particle, or any combination thereof in the non-sterile medium.

30. The system of any one of claims 25-27, wherein the first chamber comprises a manifold containing at least one pipe, wherein the manifold is in fluid communication with the second chamber via the porous membrane.

31. The system of any one of claims 25-28, further comprising a circulating flow system to remove particulate matter and debris from a surface of the porous membrane.

32. The system of claim 26, wherein the inlet port or outlet port or both are in fluid communication with a bioreactor.

33. The system of claim 31, wherein the system is essentially a closed system.

34. The system of any one of claims 25-30; wherein the system is an open system.

35. The system of claim 25, wherein the porous membrane has a pore size of less than about 220 nm.

36. The system of claim 31, wherein the porous membrane has a molecular weight cut-off (MWCO) of less than 500 kDa. 22 95815355.1Attorney Docket No.121430-791899 37. The system of any one of claims 25-35, wherein the porous membrane is comprised of polyvinylidene difluoride (PVDF), fluoropolymer, cellular acetate, composite, polyethersulfone (PES), Polyacrylonitrile or a combination thereof.

38. The system of any one of claims 25-36 wherein the porous membrane is a tubular, cylindrical, capillary, spiral wound, single leaf, plate, layered, or a hollow fiber membrane or a combination thereof.

39. The system of any one of claims 25-33, further comprising a waste port configured to remove solid waste from the system.

40. The system of claim 25, wherein the non-sterile medium is sensitive to one or more of heat, radiation or chemical treatment.

41. The system of claim 39, wherein the non-sterile medium is a basal medium, spent medium, composite medium, or conditioned medium.

42. The system of claim 40, wherein the basal medium is selected from DMEM, DMEM F12, minimum essential medium, RMI 1640, serum free medium, bench stable medium, human plasma like medium, primary culture medium, or supplemented culture medium.

43. The system of claim 39, wherein the non-sterile medium comprises at least one egg protein, or a derivative, variant of fragment thereof.

44. The system of claim 42, wherein the at least one egg protein is selected from ovalbumin, ovotransferrin, ovomucoid, ovomucin or lysozyme, apovitellenin I-VI, a lipovitellin, a livetin, phosvitin, biotin binding protein or a variant or derivative thereof, or any combination thereof.

45. The system of any one of claims 42 or 43, wherein the non-sterile medium further comprises additional egg-based ingredients.

46. The system of any one of claims 40-44, wherein the non-sterile medium further comprises growth factors, amino acids, vitamins, minerals, edible surfactants, antibiotics, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, alginates, collagen, gelatin, poly(amino acids), peptides, polypeptides, fats, fibers, buffering agents or any combinations thereof. 23 95815355.1Attorney Docket No.121430-791899 47. The system of claim 25, wherein the system does not use a pressure pump for passing the non-sterile medium through the porous membrane.

48. The system of any one of claims 25-46, wherein the system does not require heat or radiation or both for sterilizing non-sterile media.

49. The system of any one of claims 25-47, wherein the system is for use in cellular agriculture applications.

50. A method for sterilizing a non-sterile medium for cellular agriculture, the method comprising, using the system of any one of claims 25-48 to obtain a sterilized medium.

51. The method of claim 49, wherein the method reduces cost of sterilization of the non-sterile medium by at least about 20%-99%. 24 95815355.1

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