Biomimetic bioreactor systems, scaffold assemblies and methods of manufacture thereof

Abstract

According to an aspect of the present disclosure, there is a bioreactor system comprising: a cell culture vessel; and a gas exchanger unit in fluid communication with the culture chamber, the gas exchanger unit comprising: a first exchange chamber configured to receive an inflow culture liquid from the cell culture vessel, a second exchange chamber configured to hold a target gas species liquid carrier comprising an amount of target gas species substantially different from an amount of target gas species in the inflow culture liquid to cause a target gas species concentration gradient between the inflow culture liquid and the target gas species liquid carrier, a wall configured to separate the first and second exchange chambers, the wall configured to be permeable to the target gas species and substantially impermeable to liquids, wherein the target gas species concentration gradient is configured to alter the amount of target gas species in the inflow culture liquid.

Description

BIOMIMETIC BIOREACTOR SYSTEMS, SCAFFOLD ASSEMBLIES AND METHODS OF MANUFACTURE THEREOFTECHNICAL FIELD

[0001] The present disclosure relates generally to bioreactor systems, bioreactor system components, scaffold assemblies, scaffolds, scaffold sheets and methods of manufacturing thereof. The bioreactor systems and bioreactor system components include but are not limited to bioreactor systems and bioreactor system components for use in the culture of adherent cells, for example for the production of food products. The scaffold assemblies, scaffolds and / or scaffold sheets include but are not limited to biocompatible scaffold assemblies, scaffolds and / or scaffold sheets for use in bioreactors for example in the cultivation of food products.BACKGROUND

[0002] The following discussion of the background is intended to facilitate an understanding of the present disclosure only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge of the person skilled in the art in any jurisdiction as at the priority date of this application.

[0003] Most food-grade or pharmaceutical-grade bioreactors (gas-lift and stirred) are only suitable for suspension cell cultures, such as for the cultivation of lymphocytes and fibroblasts that can tolerate agitated and high shear stress suspension culture conditions. Most of these bioreactors also do not allow for adherent cell culture, needed for the cultivation of myoblasts and myocytes unless a microcarrier is used. Adherent cell cultivated myoblasts and myotubes are desired for improved organoleptic properties to mimic taste and texture or be an analogue of muscle meat.

[0004] Bioreactors aimed at adherent cells are basically for the production of cell metabolites such as in the manufacturing of pharmaceuticals, rather than harvesting of the cells themselves, and therefore use inedible materials, e.g. a strip scaffold made of PET (polyethylene terephthalate). When the purpose of the production is to harvest the cells rather than the cell- secreted products, the scaffold portion of these bioreactors becomes an obstacle to obtaining cells or may even pose a risk of foreign body residues such as microplastics in the product to be consumed.

[0005] Scaffolds made of edible materials (e.g. Merk's collagen scaffold) are currently only available for very small-scale cultures, suitable for culture systems of 0.2mL-3ml_, which is less than one-thousandth of the minimum operational volume expected of a pilot plant.

[0006] Accordingly, there is no available bioreactor (and its complement scaffold) that can fully meet the needs of culturing adherent cells (e.g., myoblasts) for food use. Therefore there exists a need to meet the needs of users (e.g. cultured / cultivated meat producers), to provide reliable, convenient, and economical equipment and consumables for the culture of adherent cells, and alleviate at least one of the aforementioned problems.SUMMARY

[0007] Bioreactor systems, bioreactor system components, scaffold assemblies, scaffolds, scaffold sheets and methods of manufacturing thereof are envisaged in the present disclosure. Bioreactor systems and bioreactor system components for use in the culture of adherent cells are envisaged in the present disclosure. Mycelial scaffold sheets and methods of manufacturing the mycelial scaffold sheets are envisaged in the present disclosure.

[0008] According to an aspect of the present disclosure, there is a bioreactor system comprising: a cell culture vessel; and a gas exchanger unit in fluid communication with the culture chamber, the gas exchanger unit comprising: a first exchange chamber configured to receive an inflow culture liquid from the cell culture vessel, a second exchange chamber configured to hold a target gas species liquid carrier comprising an amount of target gas species substantially different from an amount of target gas species in the inflow culture liquid to cause a target gas species concentration gradient between the inflow culture liquid and the target gas species liquid carrier, a wall configured to separate the first and second exchange chambers, the wall configured to be permeable to the target gas species and substantially impermeable to liquids, wherein the target gas species concentration gradient is configured to alter the amount of target gas species in the inflow culture liquid.

[0009] In some configurations, the target gas species comprises one or more of oxygen (O2), carbon dioxide (CO2), hydrogen (H2) and nitrogen (N2).

[0010] In some configurations, the second exchange chamber comprises a second exchange chamber inlet to receive a gas comprising at least the target gas species for dissolution in the target gas species liquid carrier.

[0011] In some configurations, the gas comprises air, O2, CO2, H2and / or N2.

[0012] In some configurations, the second exchange chamber comprises a target gas species disperser to disperse the received target gas species and increase an interfacial area between the target gas species and the target gas species liquid carrier.

[0013] In some configurations, the first exchange chamber comprises a first exchange chamber inlet positioned at a first end portion of the gas exchanger unit, and the second exchange chamber inlet is positioned at a second end portion of the gas exchanger unit, wherein the first and second end portions are opposing end portions of the gas exchanger unit.

[0014] In some configurations, the first exchange chamber comprises a first exchange chamber outlet positioned at the second end portion, proximal to the second exchange chamber inlet.

[0015] In some configurations, the target gas species liquid carrier comprises one or more of perfluorohexane, perfluorinated naphthalene, and glutaraldehyde polymerized bovine haemoglobin aqueous solution.

[0016] In some configurations, the cell culture vessel comprises an upper outlet and a lower outlet, wherein the upper outlet is further, relative to the lower outlet, from a surface upon which the cell culture vessel is supported.

[0017] In some configurations, the bioreactor system is configured to provide a culture liquid as the inflow culture liquid to the gas exchanger unit via one or both upper and lower outlets when a volume of the culture liquid in the cell culture vessel is above a predetermined volume.

[0018] In some configurations, the bioreactor system is configured to provide the culture liquid as the inflow culture liquid to the gas exchanger unit via the lower outlet when the volume of the culture liquid in the cell culture vessel is below the predetermined volume.

[0019] In some configurations, the cell culture vessel comprises a scaffold assembly, the scaffold assembly comprising one or more scaffold sheets configured to support adherence of cells on one or more of its surfaces.

[0020] In some configurations, the one or more scaffold sheets comprises a thickness of about 10 pm to about 100 pm.

[0021] In some configurations, a 10 mm (width) by 50 mm (length) reference portion of the one or more scaffold sheets is configured to withstand a static tensile force between about 0.01 N to about 0.5 N before failure occurs. In some configurations, a 10 mm (width) by 50 mm (length) reference portion of the one or more scaffold sheets is configured to withstand a static tensile force between about 0.04 N to about 0.15 N before failure occurs.

[0022] In some configurations, a 10 mm (width) by 50 mm (length) reference portion of the one or more scaffold sheets is configured to withstand a dynamic tensile force of at least about 0.1 N before failure occurs.

[0023] In some configurations, the one or more scaffold sheets is extensible between about 5% to about 25% of its unextended length in response to a force applied to the one or more scaffold sheets. In some configurations, the one or more scaffold sheets is extensible between about 7% to about 21% of its unextended length in response to a force applied to the one or more scaffold sheets.

[0024] In some configurations, the one or more scaffold sheets comprises a Young’s Modulus of between about 3.0 MPa to about 7.0 MPa, or between about 3.5 MPa to about 6.5 MPa.

[0025] In some configurations, the one or more scaffold sheets comprises a tensile strength of between about 0.1 MPa to about 0.6MPa.

[0026] In some configurations, the one or more scaffold sheets comprises one or more mycelial scaffold sheets comprising intertwined mycelial fibres.

[0027] In some configurations, the mycelial fibres are from one or more of fungal species comprising: Volvariella volvacea (Straw Mushroom), Lentinula edodes (shiitake mushroom), Hericium erinaceus (lion's mane mushroom), Ganoderma lucidum (lingzhi mushroom / reishi mushroom), Pleurotus eryngii (king oyster mushroom), Pleurotus ostreatus (oyster mushroom), Flammulina velutipes (enoki mushroom), Agaricus bisporus (button mushroom), Grifola frondosa (maitake mushroom) and Rhizopus oligosporus.

[0028] In some configurations, the one or more scaffold sheets comprises ergothioneine. In some configurations, the ergothioneine is from a fungal mycelium used to form the mycelial scaffold sheet.

[0029] In some configurations, the one or more mycelial scaffold sheets comprises ergothioneine at a concentration of about 0.1 mg / g to about 15.0 mg / g of a dry mycelial scaffold sheet. In some configurations, the one or more mycelial scaffold sheets comprises ergothioneine at a concentration of about 0.2 mg / g to about 12.0 mg / g of a dry mycelial scaffold sheet.

[0030] According to another aspect of the present disclosure, there is a mycelial scaffold sheet comprising intertwined mycelial fibres, the mycelial scaffold sheet configured to support adhesion of one or more cells on one or more surfaces of the mycelial scaffold sheet, wherein the mycelial scaffold sheet comprises ergothioneine.

[0031] In some configurations, the ergothioneine is from a fungal mycelium used to form the mycelial scaffold sheet.

[0032] In some configurations, the mycelial scaffold sheet comprises ergothioneine at a concentration of about 0.1 mg / g to about 15.0 mg / g of the dry mycelial scaffold sheet or about 0.2 mg / g to about 12.0 mg / g of the dry mycelial scaffold sheet.

[0033] In some configurations, the mycelial scaffold sheet comprises a non-woven sheet.

[0034] In some configurations, the mycelial fibres comprise comminuted fibres from a fungal mycelium.

[0035] In some configurations, the mycelial fibres are from one or more of fungal species comprising: Volvariella volvacea (Straw Mushroom), Lentinula edodes (shiitake mushroom), Hericium erinaceus (lion's mane mushroom), Ganoderma lucidum (lingzhi mushroom / reishi mushroom), Pleurotus eryngii (king oyster mushroom), Pleurotus ostreatus (oyster mushroom), Flammulina velutipes (enoki mushroom), Agaricus bisporus (button mushroom), Grifola frondosa (maitake mushroom) and Rhizopus oligosporus.

[0036] In some configurations, the mycelial fibres are from a plurality of fungal species.

[0037] In some configurations, the mycelial scaffold sheet is devoid of material derived from animals.

[0038] In some configurations, the mycelial scaffold sheet comprises polysaccharides ranging from about 35% to about 80% of the dry weight of the mycelial scaffold sheet.

[0039] In some configurations, the mycelial scaffold sheet comprises polypeptides ranging from about 10% to about 30% of the dry weight of the mycelial scaffold sheet.

[0040] In some configurations, the mycelial scaffold sheet comprises one or more additive compounds that are positively charged when the mycelial scaffold sheet is in use.

[0041] In some configurations, the one or more additive compounds that are positively charged when the mycelial scaffold sheet is in use, is selected from a group comprising lysine and chitosan.

[0042] In some configurations, the mycelial scaffold sheet has a thickness of about 10 pm to about 100 pm, wherein optionally, the mycelial fibres comprise an average diameter of about 1 pm to about 10 pm.

[0043] In some configurations, the mycelial scaffold sheet is configured to allow one or more particles having an average molecular weight of about 100,000 Daltons or less to pass through said mycelial scaffold sheet.

[0044] In some configurations, the mycelial scaffold sheet is edible and comprises an elasticity configured to provide a sensation of chewiness.

[0045] In some configurations, a 10 mm (width) by 50 mm (length) reference portion of the mycelial scaffold sheet is configured to withstand a static tensile force between about 0.01 N to about 0.5 N before failure occurs. In some configurations, a 10 mm (width) by 50 mm (length) portion of the mycelial scaffold sheet is configured to withstand a static tensile force between about 0.04 N to about 0.15 N before failure occurs.

[0046] In some configurations, a 10 mm (width) by 50 mm (length) reference portion of the mycelial scaffold sheet is configured to withstand a dynamic tensile force of at least about 0.1 N before failure occurs.

[0047] In some configurations, the mycelial scaffold sheet is extensible between about 5% to about 25% of its unextended length in response to a force applied to the mycelial scaffold sheet. In some configurations, the mycelial scaffold sheet is extensible between about 7% to about 21% of its unextended length in response to a force applied to the mycelial scaffold sheet.

[0048] In some configurations, the mycelial scaffold sheet comprises a Young’s Modulus of between about 3.0 MPa to about 7.0 MPa, or between about 3.5 MPa to about 6.5 MPa.

[0049] In some configurations, the mycelial scaffold sheet comprises a tensile strength of between about 0.1 MPa to about 0.6M a.

[0050] According to another aspect of the present disclosure, there is a mycelial scaffold sheet having a thickness of about 10 pm to about 100 pm, the mycelial scaffold sheet comprising intertwined mycelial fibres, wherein the mycelial scaffold sheet is configured to allow one or more particles having an average molecular weight of about 100,000 Daltons or less to pass through said mycelial scaffold sheet.

[0051] In some configurations, the mycelial fibres comprise an average diameter of about 1 pm to about 10 pm.

[0052] In some configurations, the mycelial scaffold sheet is devoid of material derived from animals.

[0053] In some configurations, the mycelial fibres are from one or more of fungal species comprising: Volvariella volvacea (Straw Mushroom), Lentinula edodes (shiitake mushroom), Hericium erinaceus (lion's mane mushroom), Ganoderma lucidum (lingzhi mushroom / reishi mushroom), Pleurotus eryngii (king oyster mushroom), Pleurotus ostreatus (oyster mushroom), Flammulina velutipes (enoki mushroom), Agaricus bisporus (button mushroom), Grifola frondosa (maitake mushroom) and Rhizopus oligosporus.

[0054] In some configurations, the mycelial scaffold sheet comprises mycelial fibres from the flat mushroom species and wherein the mycelial scaffold sheet is configured to allow one or more particles having an average molecular weight of about 1 ,000 Daltons or less to pass through said mycelial scaffold sheet.

[0055] In some configurations, the mycelial scaffold sheet comprises Lentinula edodes mycelial fibres and wherein the mycelial scaffold sheet is configured to allow one or more particles having an average molecular weight of about 100,000 Daltons or less to pass through said mycelial scaffold sheet.

[0056] In some configurations, the mycelial scaffold sheet comprises a non-woven sheet. In some configurations, the mycelial fibres comprise comminuted fibres from a fungal material. The fungal material may comprise fungal mycelium. In some configurations, the mycelial scaffold sheet comprises polysaccharides ranging from about 35% to about 80% of the dry weight of the mycelial scaffold sheet.

[0057] In some configurations, the mycelial scaffold sheet comprises polypeptides ranging from about 10% to about 30% of the dry weight of the mycelial scaffold sheet.

[0058] In some configurations, the mycelial scaffold sheet comprises one or more additive compounds that are positively charged when the mycelial scaffold sheet is in use. The one or more additive compounds that are positively charged when the mycelial scaffold sheet is in use, may be selected from a group comprising lysine and chitosan. The lysine may be a polylysine.

[0059] In some configurations, the mycelial scaffold sheet is edible and comprises an elasticity configured to provide a sensation of chewiness.

[0060] In some configurations, a 10 mm (width) by 50 mm (length) reference portion of the mycelial scaffold sheet is configured to withstand a static tensile force between about 0.01 N to about 0.5 N before failure occurs. In some configurations, a 10 mm (width) by 50 mm (length) reference portion of the mycelial scaffold sheet is configured to withstand a static tensile force between about 0.04 N to about 0.15 N before failure occurs.

[0061] In some configurations, a 10 mm (width) by 50 mm (length) reference portion of the mycelial scaffold sheet is configured to withstand a dynamic tensile force of at least about 0.1 N before failure occurs.

[0062] In some configurations, the mycelial scaffold sheet is extensible between about 5% to about 25% of its unextended length in response to a force applied to the mycelial scaffold sheet. In some configurations, the mycelial scaffold sheet is extensible between about 7% to about 21% of its unextended length in response to a force applied to the mycelial scaffold sheet.

[0063] In some configurations, the mycelial scaffold sheet comprises a Young’s Modulus of between about 3.0 MPa to about 7.0 MPa, or between about 3.5 MPa to about 6.5 MPa. In some configurations, the mycelial scaffold sheet comprises a tensile strength of between about 0.1 MPa to about 0.6MPa.

[0064] According to another aspect of the present disclosure, there is a scaffold assembly for use in a bioreactor, the scaffold assembly comprising a float and a flexible biocompatible scaffold extending from the float.

[0065] In some configurations, at least a portion of the flexible biocompatible scaffold is formed from plant and / or fungal material.

[0066] In some configurations, at least a portion of the flexible biocompatible scaffold is formed from edible fibres. The edible fibres may comprise edible mycelial fibres.

[0067] In some configurations, the biocompatible scaffold comprises mycelial fibres from one or more of fungal species comprising: Volvariella volvacea (Straw Mushroom), Lentinula edodes(shiitake mushroom), Hericium erinaceus (lion's mane mushroom), Ganoderma lucidum (lingzhi mushroom / reishi mushroom), Pleurotus eryngii (king oyster mushroom), Pleurotus ostreatus (oyster mushroom), Flammulina velutipes (enoki mushroom), Agaricus bisporus (button mushroom), Grifola frondosa (maitake mushroom) and Rhizopus oligosporus.

[0068] In some configurations, the flexible biocompatible scaffold comprises one or more scaffold sheets. In some configurations, the one or more scaffold sheets comprise one or more mycelial scaffold sheets according to an aspect of the present disclosure.

[0069] In some configurations, the scaffold assembly comprises a plurality of scaffold sheets connected together by one or more connector strips of plant and / or fungal material.

[0070] In some configurations, the one or more connector strips are woven through a central portion of at least one scaffold sheet.

[0071] In some configurations, the one or more connector strips comprises the same material as the scaffold sheets.

[0072] In some configurations, a portion of a scaffold sheet is spaced apart from a portion of another scaffold sheet, wherein the portions are at or proximal to the one or more connector strips.

[0073] In some configurations, the one or more scaffold sheets comprises at least one channel extending through the scaffold sheet.

[0074] In some configurations, the float comprises a density less than a density of a liquid that it is configured to float in or on.

[0075] In some configurations, the float is arranged at a top portion of the scaffold assembly, such that when the scaffold assembly is in use, the float is configured to float on, at or proximal a surface of a or the liquid, and the flexible biocompatible scaffold is configured to suspend within the liquid.

[0076] According to another aspect of the present disclosure, there is a buoyant scaffold assembly comprising a biocompatible scaffold for supporting cells, wherein the biocompatible scaffold is configured to move between a contracted configuration and an expanded configuration based on a volume of a liquid in a bioreactor.

[0077] In some configurations, the biocompatible scaffold comprises a plurality of scaffold sheets connected together by one or more connector strips. One or more scaffold sheets may comprise one or more mycelial scaffold sheets according to an aspect of the present disclosure.

[0078] In some configurations, a portion of a scaffold sheet is spaced apart from a portion of another scaffold sheet, wherein the portions are at and / or proximal to the one or more connector strips.

[0079] In some configurations, at least one connector strip is flexible to vary a distance between neighbouring scaffold sheets when in use.

[0080] In some configurations, at least one connector strip comprises at least one bend along its length to vary a distance between neighbouring scaffold sheets when in use.

[0081] In some configurations, at least a portion of the biocompatible scaffold is formed from edible mycelial fibres.

[0082] In some configurations, the biocompatible scaffold comprises mycelial fibres from one or more of fungal species comprising: Volvariella volvacea (Straw Mushroom), Lentinula edodes (shiitake mushroom), Hericium erinaceus (lion's mane mushroom), Ganoderma lucidum (lingzhi mushroom / reishi mushroom), Pleurotus eryngii (king oyster mushroom), Pleurotus ostreatus (oyster mushroom), Flammulina velutipes (enoki mushroom), Agaricus bisporus (button mushroom), Grifola frondosa (maitake mushroom) and Rhizopus oligosporus.

[0083] According to another aspect of the present disclosure, there is a method of forming a biocompatible scaffold sheet comprising the steps of: comminuting plant and / or fungal material; forming a suspension in a liquid from the comminuted plant and / or fungal material; spreading the suspension out on a support; and micro piercing the spread suspension to cause fibres in the plant and / or fungal material to intertwine.

[0084] In some configurations, the method further comprises adding one or more additive compounds to the suspension, wherein the one or more additive compounds are configured to be positively charged when the biocompatible scaffold sheet is in use. The one or more additive compounds may be selected from a group comprising lysine and chitosan. The lysine may be a polylysine.

[0085] In some configurations, the support comprises a mesh and the method comprises spreading the suspension out on the mesh to achieve a thickness of the suspension of about 0.5 mm to about 2 mm.

[0086] In some configurations, the method further comprises compressing and drying the spread suspension.

[0087] In some configurations, the crushed plant and / or fungal material comprises edible mycelial fibres.

[0088] In some configurations, the step of forming the suspension further comprises mixing the crushed plant and / or fungal material with one or more of a polysaccharide (such as pea starch) and a fungal and / or plant protein (such as a Ganoderma lucidum protein, a mushroom protein and a soy protein).

[0089] In some configurations, the fungal material is from one or more of fungal species comprising: Volvariella volvacea (Straw Mushroom), Lentinula edodes (shiitake mushroom), Hericium erinaceus (lion's mane mushroom), Ganoderma lucidum (lingzhi mushroom / reishi mushroom), Pleurotus eryngii (king oyster mushroom), Pleurotus ostreatus (oyster mushroom), Flammulina velutipes (enoki mushroom), Agaricus bisporus (button mushroom), Grifola frondosa (maitake mushroom) and Rhizopus oligosporus.

[0090] According to another aspect of the present disclosure, there is a biocompatible scaffold sheet formed from a method according to an aspect of the present disclosure, the biocompatible scaffold comprising mycelial fibres intertwined by the step of micro-piercing.

[0091] In some configurations, the biocompatible scaffold sheet comprises a thickness of about 10 pm to 100 pm.

[0092] In some configurations, the biocompatible scaffold sheet is devoid of material derived from animals.

[0093] According to another aspect of the present disclosure, there is a scaffold assembly for use in a bioreactor, the scaffold assembly comprising one or more mycelial scaffold sheets according to an aspect of the present disclosure.

[0094] According to another aspect of the present disclosure, there is a bioreactor system comprising one or more mycelial scaffold sheets according to an aspect of the present disclosure.

[0095] According to another aspect of the present disclosure, there is a bioreactor for use in the cultivation of a food product, the bioreactor comprising a mycelial scaffold sheet according to an aspect of the present disclosure or a biocompatible scaffold sheet according to an aspect of the present disclosure, wherein the mycelial scaffold sheet or biocompatible scaffold sheet is edible and is configured to support at least partially adherent animal cells to form the food product.

[0096] According to an aspect of the present disclosure, there is a bioreactor system comprising: a cell culture vessel; and a metabolite management device in fluid communication with the culture cell vessel, the metabolite management device comprising: a first chamber comprising a first electrode, the first chamber configured to receive a first buffer fluid; a second chamber comprising a second electrode, the second chamber configured to receive a second buffer fluid; and a third chamber configured to receive an inflow culture fluid from the cell culture vessel, the third chamber is separated from each of the first and second chambers by a first and second semi-permeable wall respectively, wherein the first or second electrode is configured to be charged to form a positive electrode and the other of the first or second electrode is configured to be charged to form a negative electrode, the positive and negative electrodes configured to apply an electric field to at least the inflow culture fluid in the third chamber to cause positively charged components of the inflow culture fluid to move towards the negative electrode and negatively charged components of the inflow culture fluid to move towards the positive electrode, and wherein the first and second semi-permeable walls are configured to permit positively and negatively charged components less than a predetermined size to pass from the third chamber to the first and / or second chambers.

[0097] In some configurations, components of the inflow culture fluid that are capable of passing through the first and second semi-permeable walls are configured to concentrate in the first and second buffer fluids.

[0098] In some configurations, the first and / or second semi-permeable walls are removable from the metabolite management device. In some configurations, the first and / or second semi- permeable walls comprise a semi-permeable membrane.

[0099] In some configurations, the first and / or second electrode comprises a conductive material. The conductive material may comprise one or more of a metal and graphite.

[0100] In some configurations, the first chamber comprises a first chamber inlet configured to receive the first buffer fluid and a first chamber outlet configured to discharge the first buffer fluid; the second chamber comprises a second chamber inlet configured to receive the second buffer fluid and a second chamber outlet configured to discharge the second buffer fluid; and the third chamber comprises a third chamber inlet configured to receive the inflow culture fluid from the cell culture vessel and a third chamber outlet configured to discharge an outflow culture fluid.

[0101] In some configurations, flow of the outflow culture fluid out of the third chamber outlet is under the effect of gravity.

[0102] In some configurations, the bioreactor system further comprises a culture fluid pump configured to pump the inflow culture fluid to the third chamber via the third chamber inlet.

[0103] In some configurations, the bioreactor system further comprises one or more buffer pumps configured to pump the first and / or second buffer fluids from one or more buffer fluid sources to the first and / or second chambers via the respective first and second chambers inlets. The first and second buffer fluids may be the same buffer fluid.

[0104] In some configurations, the electric field is applied to at least the inflow culture fluid in the third chamber when the culture fluid pump has pumped the inflow culture fluid to the third chamber.

[0105] In some configurations, the first, second and third chamber outlets are configured to operate between a closed and open configuration, wherein when the electric field is applied, the first, second and third chamber outlets are in the closed configuration. In some configurations when the electric field is stopped or absent, the outlets may be in the open configuration.

[0106] According to another aspect of the present disclosure, there is a bioreactor system comprising: cell culture vessel; a bioreactor system component; and a pump fluidly connecting the cell culture vessel and the bioreactor system component, the pump comprising a compressible pump chamber, a pump chamber inlet and a pump chamber outlet, wherein the compressible pump chamber is operable between a constricted configuration and a dilated configuration,wherein operation of the pump chamber from the constricted configuration to the dilated configuration causes movement of an inflow culture fluid from the cell culture vessel or the bioreactor system component into the pump chamber via the pump chamber inlet, and wherein operation of the pump chamber from the dilated configuration to the constricted configuration causes movement of an outflow fluid out of the pump chamber to the cell culture vessel or the bioreactor system component via the pump chamber outlet.

[0107] In some configurations, the pump comprises two or more rigid plates moveable relative to each other and the compressible pump chamber is arranged between the rigid plates, wherein movement of the rigid plates causes the operation of the pump chamber between the constricted and dilated configurations.

[0108] In some configurations, the pump chamber inlet and / or pump chamber outlet comprises a one-way valve.

[0109] In some configurations, the pump chamber inlet comprises the one-way valve, and wherein the one-way valve is configured to remain at least partially open for a reverse flow of a portion of the outflow fluid through the pump chamber inlet when the pump chamber is operated from the dilated configuration to the constricted configuration.

[0110] In some configurations, the bioreactor system further comprises a filter to filter the inflow fluid entering the pump chamber via the pump chamber inlet.

[0111] In some configurations, the bioreactor system further comprises a first fluid channel and a second fluid channel configured to receive the outflow fluid from the pump chamber outlet, wherein the first fluid channel is configured to provide a first portion of the outflow fluid to the cell culture vessel or the bioreactor system component, and wherein the second fluid channel is configured to provide a second portion of the outflow fluid to a portion of the bioreactor system upstream of the pump chamber inlet.

[0112] In some configurations, the second portion of the outflow fluid comprises a lower flow rate relative to the first portion of the outflow fluid.

[0113] According to another aspect of the present disclosure, there is a bioreactor system comprising: a cell culture vessel; anda media incubation device in fluid communication with the culture chamber, the media incubation device comprising an incubation chamber configured to receive an inflow culture fluid, the incubation chamber comprising an immobilisation structure configured to support an immobilised enzyme, wherein the immobilised enzyme is configured to enzymatically sterilize the inflow culture fluid before discharging the inflow culture fluid as an outflow culture fluid to the cell culture vessel.

[0114] In some configurations, the incubation chamber comprises an incubation chamber inlet to receive the inflow culture fluid and an incubation chamber outlet to discharge the outflow culture fluid, the incubation chamber inlet and / or outlet comprising a filter.

[0115] In some configurations, the media incubation device comprising a heater or cooler configured to heat or cool the inflow culture fluid.

[0116] In some configurations, the media incubation device is configured to incubate the inflow culture fluid for a predetermined amount of time for temperature control of the inflow culture fluid before discharging the inflow culture fluid as the outflow culture fluid.

[0117] In some configurations, the immobilisation structure comprises one or more baffles.

[0118] According to another aspect of the present disclosure, there is a bioreactor system comprising: a chamber; and a chamber cover configured to seal the chamber from an ambient environment, wherein a wall of the chamber cover is spaced from a wall of the chamber to form a protective fluid cavity between the chamber and chamber cover to permit containment of a protective fluid to minimize or prevent contamination of the chamber and / or its contents from the ambient environment.

[0119] In some configurations, the chamber cover comprises one or more openings to allow one or more bioreactor system components or portions thereof to extend through.

[0120] In some configurations, the chamber cover comprises a first cover portion and a second cover portion, each of the first and second cover portions comprising a recess and each portion removably connectable with the other to form a chamber cover cavity with their respective recesses, wherein the chamber cover cavity is configured to house the chamber.

[0121] In some configurations, the protective fluid comprises one or more of ozone and air.

[0122] In some configurations, the chamber cover is configured to maintain the chamber at a desired temperature.

[0123] Other aspects and features of the present disclosure will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the present disclosure in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0124] In the figures, which illustrate, by way of non-limiting examples only, embodiments of the present disclosure,

[0125] [Fig. 1]: A schematic diagram of an embodiment of a bioreactor system with an embodiment of a scaffold assembly of the present disclosure.

[0126] [Fig. 2]: A perspective view of an illustration of an embodiment of a scaffold assembly of the present disclosure.

[0127] [Fig. 3]: A perspective view of an illustration of an embodiment of a scaffold assembly of the present disclosure, where the scaffold sheets are shown as translucent.

[0128] [Fig. 4]: An illustration of an embodiment of a scaffold assembly of the present disclosure in a contracted configuration (A) and an expanded configuration (B).

[0129] [Fig. 5]: A photograph of an embodiment of a scaffold sheet of the present disclosure.

[0130] [Fig. 6]: Analytic data of an embodiment of a scaffold of the present disclosure: (A) GC / MS (Gas chromatography / mass spectrometry) lipid profiles of an embodiment of a scaffold of the present disclosure; (B) LC / MS (Liquid chromatography-mass spectrometry) water soluble vitamin profiles of an embodiment of a scaffold of the present disclosure; (C) ATR-FTIR (Attenuated total reflectance methodology for Fourier transform infrared spectroscopy) spectrum of an embodiment of a scaffold of the present disclosure.

[0131] [Fig. 7]: A graph showing changes in strength of an embodiment of a mycelium scaffold sheet of the present disclosure under various media conditions during submersion.

[0132] [Fig. 8]: A graph of tensile tests of an embodiment of a mycelium scaffold sheet of the present disclosure.

[0133] [Fig. 9]: A schematic diagram of an embodiment of a bioreactor system of the present disclosure.

[0134] [Fig. 10]: A schematic diagram of an embodiment of a compression reflux liquid pump of the present disclosure.

[0135] [Fig. 11]: Schematic diagrams of an embodiment of a gas exchange vessel of the present disclosure.

[0136] [Fig. 12]: A schematic diagram of an embodiment of a metabolite separation device of the present disclosure with a wall of the device removed to show the internal structure of said metabolite separation device.

[0137] [Fig. 13]: A perspective view of a schematic diagram of an embodiment of a sterilization jacket of the present disclosure.

[0138] [Fig. 14]: A schematic cross-section diagram of an embodiment of a media incubation unit of the present disclosure.DETAILED DESCRIPTION

[0139] Throughout this document, unless otherwise indicated to the contrary, the terms “comprising”, “consisting of’, “having” and the like, are to be construed as non-exhaustive, or in other words, as meaning “including, but not limited to”.

[0140] Furthermore, throughout the document, unless the context requires otherwise, the word “include” or variations such as “includes” or “including” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0141] Throughout the description, it is to be appreciated that the term and its plural form include.

[0142] Throughout the description, it is to be appreciated that the term and its plural form can include.

[0143] Throughout the description, it is to be appreciated that the term or ‘-s’ include.

[0144] Unless defined otherwise, all other technical and scientific terms used herein have the same meaning as is commonly understood by a skilled person to which the subject matter herein belongs.

[0145] [Fig. 1] provides a schematic diagram of a bioreactor system 1000 according to an embodiment of the present disclosure and its components. The bioreactor system 1000 may be used for an adherent cell culture. The bioreactor system 1000 may be used in the culture of cultivated food products (e.g. meat products), and may be suitable not only for adhesiondependent cell types (e.g. myoblasts), but also for culturing partially adherent cell types (e.g. fibroblasts).

[0146] The bioreactor system 1000 may also be used for adherent cell cultures for non-food purposes, such as cell cultures for the purpose of collecting secreted products (e.g., fibroblast growth factor), or for the production of human or other animal tissue for medical, veterinary, or other pharmaceutical and research and development purposes.

[0147] The bioreactor system 1000 comprises a cell culture vessel 1100, one or more liquid pumps 1300, a gas exchange vessel 1400, a temperature control module 1500 and one or more conduits 1600 connecting the various components of the bioreactor system 1000. As used in the present disclosure, the term “cell culture vessel” includes a bioreactor that is used to culture cells. Therefore, the term “cell culture vessel” may be used interchangeably with the term “bioreactor” in the present disclosure. Components of bioreactor system 1000 may be collectively referred to as bioreactor system components in the present disclosure. The bioreactor system 1000 further comprises a scaffold assembly 1200 housed in cell culture vessel 1100. The bioreactor 1100 may optionally include a sterilization jacket (not shown). The bioreactor 1100 may comprise one or more auxiliary components such as sensors (e.g. oxygen (O2) sensor 1101, carbon dioxide (CO2) sensor 1103, pH sensor 1102, and temperature sensor 1104), auxiliary pumps (e.g. sodium hydroxide (NaOH) pump 1403 and air pump 1401), gas sources (e.g. CO2 gas source 1402) and control devices (e.g. temperature control module 1500 and control unit 1700). The NaOH pump is configured to achieve a desired pH in the culture medium (e.g. pH 7) in the bioreactor system 1000. A desirable pH range may be pH 6.8-7.6. The sensors may sense one or more parameters of a fluid (e.g. a liquid or a gas) in one or more vessels or other components connecting the vessels. One or more control devices may control one or more components of the bioreactor system 1000. One or more control devices may receive a signal from the one or more sensors as feedback control of one or more components of the bioreactor. The one or more control devices may be communicatively coupled (e.g. wired or wirelessly) with one or more components of the bioreactor system 1000. For example, when the pH sensor 1102 measures that the pH of afferent culture medium entering cell culture vessel 1100 is lower than a minimum threshold of a desirable pH range, the pH sensor 1102 communicates this pH measurement to the control unit 1700 which will in turn communicate with the NaOH pump 1403 to dispense NaOH into the culture medium in the gas exchange vessel 1400 to increase the pH to a pH within the desirable pH range.

[0148] In some examples, the bioreactor system 1000 includes other bioreactor components. For example, the bioreactor system 100 may include metabolite separation unit 2800 as described in bioreactor system 2000. Components of the bioreactor system 1000 may also be arranged in the same or similar manner as another embodiment of a bioreactor system of the present disclosure. In some examples, components of bioreactor system 1000 are arranged to provide for one flow path as shown in [Fig 1] and as described below. In some examples, components of the bioreactor system 1000 are arranged to provide for two or more flow paths, e.g. flow paths A and B as described in relation to bioreactor system 2000 in the present disclosure.

[0149] The cell culture vessel 1100 can be made of glass, stainless steel with or without a window, or plastics that are food-safe and resistant to high-temperatures during sterilization or retort. The cell culture vessel 1100 comprises a cover 1105 with ports. The cover 1105 may be a removable cover. The ports may be configured to position one or more components such as sensors and conduits (for transport of culture medium). The cell culture vessel 1100 comprises an outlet 1107. The outlet 1107 is positioned on the cover 1105 and may be provided in the middle of the cover 1105. The cell culture vessel 1100 comprises an inlet 1108 in a base portion of one side of the vessel 1100. It will be appreciated that the outlet 1107 and inlet 1108 may be arranged differently on the cell culture vessel 1100, for example, both outlet 1107 and inlet 1108 may be arranged on the cover 1105, or both outlet 1107 and inlet 1108 may be arranged on the side of the cell culture vessel 1100. In some examples, there is more than one outlet 1107 and / or more than one inlet 1108. In some examples, the inlet 1108 may be positioned at a distance from the outlet 1107, such that culture medium flowing into the cell culture vessel 1100 via the inlet 1108 may flow through a substantial portion of the cell culture vessel 1110 before exiting the outlet 1107. In some examples, one or both outlet 1107 and inlet 1108 may be provided with a conduit that extends into the cell culture vessel 1100. In some examples, the lengths of the conduits that extend into the cell culture vessel 1100 from the outlet 1107 and / or inlet 1108 may be the same or different. For example, if both outlet 1107 and inlet 1108 are provided on the cover 1105, the conduit extending from inlet 1108 though which culture medium enters the cell culture vessel 1100 may extend closer to the base of the cell culture vessel 1100 relative to the conduit extending from outlet 1107 though which culture medium exits cell culture vessel 1100. The cover 1105 can be fitted with a variety of commercially available sensors (e.g. pH sensor 1102, temperature sensor 1104, dissolved oxygen detector 1101 , CO2 sensor 1103 etc.) to monitor one or more parameters of the cell culture in the cell culture vessel 1100, for example to monitor cell growth. The cell culture vessel 1100 may have an inoculation port (not shown) and a feeder port 1109 for adding culture medium. With reference to cell culture vessel 1100, efferent culture medium is pumped out of the upper part of the vessel 1100 via the outlet 1107 by a liquid pump 1300 through a conduit 1600 into the gas exchange vessel 1400, while oxygen-rich afferent culture medium isreturned to the cell culture vessel 1100 via inlet 1108 from the bottom of the vessel 1100. The cell culture vessel 1100 can be insulated on its outside, but the insulation may need to include a transparent window section for inspection during the cell cultivation process. One or more filters 1106 may be provided to the cell culture vessel 1100, e g. at an end of a conduit 1600 to prevent or minimize cells in the cell culture vessel 1100 from flowing out of the cell culture vessel 1100 when culture medium is pumped out of the cell culture vessel 1100.

[0150] The gas exchange vessel 1400 enriches oxygen and regulates the carbon dioxide content of the cultivation media (i.e. culture medium). The gas exchange vessel 1400 may also enrich other target gas species such as nitrogen and hydrogen in the culture medium. The gas exchange vessel 1400 is smaller than the cell culture chamber 1100 and can be made of glass, stainless steel with or without a window, or food-safe and autoclavable plastics. The gas exchange vessel 1400 is described below in more detail with respect to gas exchange vessel 2400 ([Fig. 11]). Briefly, gas exchange vessel 1400 comprises a cavity configured to be filled with a target gas species liquid carrier comprising an amount of target gas species substantially different from an amount of target gas species in the inflow culture liquid received by the gas exchange vessel, to cause a target gas species concentration gradient between the inflow culture liquid and the target gas species liquid carrier. The target gas species concentration gradient is configured to alter the amount of target gas species in the inflow culture liquid. Air, oxygen (O2), carbon dioxide (CO2), nitrogen (N2) and / or hydrogen (H2) is provided to the target gas species liquid carrier from one or more gas sources, e.g. ambient air pumped into the gas exchange vessel 1400 via air pump 1401 and CO2 is supplied to the gas exchange vessel 1400 from a CO2 gas source 1402. Other gas sources such as O2, N2 and H2 gas sources are also envisaged in the present disclosure. The gas exchange vessel 1400 may comprise aeration devices (e g. aeration disks, aeration tubes, etc ). The aeration devices may evenly distribute gases introduced into the target gas species liquid carrier of the gas exchange vessel 1400 and / or generate fine gas bubbles to increase surface area and increase dissolution of the gases into the target gas species liquid carrier. Gases may be introduced at the base of the gas exchange vessel 1400 and bubbled through the target gas species liquid carrier. Regulation of gases in a vessel separate from the cell culture vessel 1100 can reduce impact of the gases on the growth and yield of the cells and / or reduce or prevent damage to the scaffold 1200 in the cell culture vessel 1100. For example, bubbles caused by introduced gases can cause damage to cultivated cells and / or the scaffold 1200 in the cell culture vessel 1100.

[0151] Movement of culture medium in bioreactor system 1000 is driven by the one or more liquid pumps 1300. The bioreactor system 1000 may comprise a plurality of liquid pumps 1300 such that one or more liquid pumps 1300 may function as redundancy in the event of a failure in one or more other liquid pumps 1300. Where the bioreactor system 1000 comprises a plurality ofliquid pumps 1300, the liquid pumps 1300 may be the same or different pumps and / or may be configured to drive the same or different volumetric flow rate. In the bioreactor system 1000 of [Fig. 1], the one or more liquid pumps 1300 comprises one or more peristaltic pumps. In some examples, the one or more liquid pumps 1300 comprises one or more diaphragm pumps (e.g. compression reflux liquid pump 2300 as described below).

[0152] The cell culture vessel 1100 and gas exchange vessel 1400 can be provided with a sterilization jacket. The jacket may be a jacket according to an embodiment of the present disclosure. The jacket may form an air pocket between the jacket and the outer wall of the cell culture vessel 1100 and / or gas exchange vessel 1400. The jacket can be made of soft or hard plastic, resembling a cylinder split in the middle, with a sealing structure made of soft, temperature-resistant material (e.g. silicone, foamed silicone, rubber, thermoplastic polyurethane (TPU), etc.) with symmetrical grooves at an opening. The jacket may have a cross-sectional shape that corresponds with the cross-sectional shape of the vessel it contains. The overall shape of its closure may be a cylinder, cube, or other polygonal prism. When in use, the cell culture vessel 1100 or gas exchange vessel 1400 is placed into the jacket through the opening, the conduits 1600 connected to the vessel 1100, 1400 are adjusted into the grooves in the opening of the jacket, and the jacket is closed and secured with a locking clip. Seal the recesses of the sealing structure redundantly with plugs of matching diameter, i.e. one or more redundant recesses of the jacket may be sealed with plugs of matching diameter.

[0153] The jacket may comprise conduits for connection to an ozone generator or for ventilation of filtered air. The conduits may be closed after use. The space between the jacket and the vessel 1100, 1400 may act as a buffer space so that microorganisms from the external environment cannot directly invade the bioreactor, reducing the probability of bioreactor contamination.

[0154] The cell culture vessel 1100 includes a scaffold assembly 1200 to support adhesion of cells. In some examples, the scaffold assembly 1200 comprises one or more substantially planar platforms arranged, for example, in a substantially horizontal, vertical or a combination thereof manner, and which are configured to support adhesion of cells. In some examples, the platforms comprise curves. In some examples, profiles of the platforms are dependent on fluid dynamics of the cell culture medium in which the scaffold assembly 1200 is placed. The one or more platforms may be rigid or flexible. The one or more platforms may be porous. The scaffold assembly 1200 may comprise a plurality of platforms permanently or removably connected to one another. The scaffold assembly 1200 may be a scaffold assembly according to an embodiment of the present disclosure. The scaffold assembly 1200 may comprise any suitable material, for example, metals, polymers and ceramics. The polymers may be inert or reactive (also known as biodegradable)polymers. The scaffold assembly 1200 may comprise a biocompatible material. The biocompatible material may be an edible material, for example a material derived from plants and / or fungi. The scaffold assembly 1200 may comprise a multi-layered scaffold structure, for example multiple scaffold sheets (i.e. platforms), that increase the surface area for adhesion and exposure of the adhered cells to the cultivation medium in the cell culture vessel. The scaffold structure may be a scaffold according to an embodiment of the present disclosure. One of more scaffold sheets may be a scaffold sheet according to an embodiment of the present disclosure. The scaffold assembly 1200 may be provided in a fixed or unfixed position in the cell culture vessel 1100. The scaffold assembly 1200 may be connected, for example removably connected to a wall of the cell culture vessel 1100.

[0155] Scaffold Sheet, Scaffold and Scaffold Assembly

[0156] [Fig. 2] to [Fig. 8] relate to embodiments of a scaffold sheet, scaffold and scaffold assembly of the present disclosure. While the embodiments of the scaffold sheet, scaffold and scaffold assembly are described below with respect to bioreactor system 1000, it will be appreciated that these embodiments may be used in any other bioreactor system according to an embodiment of the present disclosure

[0157] [Fig. 2], [Fig. 3] and [Fig. 4] show a scaffold assembly 200 according to an embodiment of the present disclosure and [Fig. 5] shows a scaffold sheet 220 according to an embodiment of the present disclosure. Scaffold assembly 200 comprises a scaffold 210 (also referred to as a scaffold structure in the present disclosure). The scaffold assembly 200 may comprise or be formed from plant and / or fungal fibres. The scaffold assembly 200 comprises a buoyant scaffold assembly 200. The biocompatible scaffold 210 comprises a flexible scaffold 210. The biocompatible scaffold 210 includes one or more scaffold sheets 220, an example of which is shown in [Fig. 5], The scaffold sheet 220 may be flexible. A portion of the biocompatible scaffold 210 may comprise or be formed from plant and / or fungal fibres. The fibres may be inedible or edible. The fibres may be mycelial fibres. One or more scaffold sheets 220 may be formed of edible mycelial fibres. The scaffold assembly may include a plurality of scaffolds sheets 220.

[0158] In an example, multiple layers of scaffold sheets 220 are attached in series to a float 231 , which is not shown in [Fig 2 ] and [Fig. 3] but is shown in [Fig. 1], [Fig. 4] and [Fig 9], The float 231 is buoyant in the cell culture medium. The float 231 may have a lower density than the liquid cultivation medium in the bioreactor, or may contain a cavity filled with air, so that, when in use, the float 231 can float in the cultivation medium (e.g. the liquid surface) and the multiple layers of scaffold sheets 220 are suspended within the cultivation medium, e.g. below the liquid surface or meniscus of the liquid cultivation medium. Therefore, the scaffold assembly 200 maycomprise a scaffold 210 and a float 231, where the scaffold 210 extends from the float 231. The suspended scaffold sheets 220 may drape towards the base of the cell culture vessel 1100 under the effect of gravity. Portions of the suspended scaffold sheets 220 may move based on the fluid dynamics of the cultivation medium when in use. In an example, the end portions 223 of suspended scaffold sheets 220 move based on the fluid dynamics of the cultivation medium when in use. The float 231 may be positioned at the top of the scaffold assembly 200, such that when the scaffold assembly 200 is in use, the float 231 is configured to float on, at or proximal the surface of the liquid cultivation medium, and the scaffold 210 is configured to suspend within the liquid cultivation medium. In some examples, the float 231 is configured to float within the liquid cultivation medium at a predetermined distance from the surface of the liquid cultivation medium. The float 231 may be made of metal. In some examples, the buoyant scaffold assembly 200 may not comprise a float 231 , and in such examples, a portion of scaffold 210 may be configured with a certain buoyancy (or density) and is capable of floating in the cell culture medium.

[0159] In an example, the scaffold assembly 200 comprises a buoyant scaffold assembly comprising a scaffold 210 configured to move between a contracted configuration and an expanded configuration based on a volume of a liquid in a bioreactor as shown in [Fig. 4]. [Fig. 4A] shows the scaffold assembly 200 in a contracted configuration and [Fig. 4B] shows the scaffold assembly 200 in an expanded configuration. The ability to move between these two configurations allows the scaffold assembly 200 to be positioned early within the bioreactor 1100 when the volume of the liquid in the bioreactor 1100 is low, e.g. during cell inoculation ([Fig. 4A]), so that it does not need to only be added when the volume of liquid is increased to a minimal amount, thereby minimizing contamination risks. In the contracted configuration ([Fig. 4A]), the scaffold sheets 220 of the scaffold 210 are located close to each other and may be positioned adjacent to and in contact with one another. In the expanded configuration ([Fig. 4B]), the scaffold sheets 220 are spaced apart to allow cells and media to flow between the scaffold sheets 220 to facilitate cell adhesion on the scaffold sheets 220. The flexibility of the scaffold sheets 220 allow them to drape towards the base of the cell culture 1100 under the effect of gravity in the expanded configuration.

[0160] In an example, the scaffold 210 comprises a portion formed of plant material, e g. the scaffold sheet may not be mycelial scaffold sheet 220 but another scaffold sheet formed of plant fibres. In an example, the scaffold 210 comprises a formed of plant and fungal material. The scaffold assembly 200 comprises stacked scaffold sheets 220 that are connected together using one or more connector strips 230 of plant and / or fungal material (e.g. mycelium) woven through the centre of the scaffold sheets 220. As shown in [Fig. 2] and [Fig. 3], the connector strips 230 are woven through a central portion of at least one mycelial scaffold sheet 220. In some examples, the connector strips 230 are woven through a side portion of at least one mycelial scaffold sheet220. The one or more connector strips 230 of plant and / or fungal material may be formed of the same material as the scaffold sheet 220. In some examples, the one or more connector strips 230 may comprise or be formed of other material, such as metals, polymers and ceramics. The one or more connector strips 230 may be formed of an inert material. The one or more connector strips 230 of plant and / or fungal material that connects the scaffold sheets 220 may be configured to maintain space between the scaffold sheets 220 for culture media flow and animal cell growth. A portion of a scaffold sheet 220 may therefore be spaced apart from a portion of another scaffold sheet 220, wherein the portions are at or proximal to the one or more connector strips 230. The scaffold sheets 220 may therefore be positioned at predetermined distances from one another along the length of the one or more connector strips 230. These predetermined distances may be the same or different between scaffold sheets 220. The scaffold sheets 220 may comprise one or more channels 221 extending through each sheet to allow for improved hydrodynamics, allowing culture media and its nutrients and oxygen to flow. [Fig. 3] provides another perspective view of the scaffold assembly 200 where the scaffold sheets 220 are shown as translucent which clearly shows the one or more interwoven connector strips 230 of plant and / or fungal material. While [Fig. 3] shows the connector strips 230 as being thicker than the scaffold sheets 220, the connector strips 230 may be thicker than, thinner than or have the same thickness as the scaffold sheets 220.

[0161] When the scaffold assembly 200 is in use, at the early stage of inoculation, a lower liquid cultivation medium volume can be set, such that the liquid level is lower, and the scaffold sheets 220 (or at least its end portions thereof) are stacked in greater proximity to each other at or near the bottom of the cell culture vessel 1100 (contracted configuration). When the cultivation medium’s liquid level rises in the mid-term and later stages of incubation due to the addition of cultivation media in a cultivation process, the scaffold sheets 220 can gradually unfold from partially to completely with the rising position of the float (expanded configuration). This may be advantageous because the cell culture vessel 1100 does not need to be filled with cultivation medium to its maximum capacity at the earliest stage culture media which can have a considerable impact on cost in large scale cultivation, and the inoculum volume does not need to be significantly large as this can avoid a long delay in the cell growth curve. The scaffold assembly 200 is therefore self-adjusting in its height and lamellar distribution at different stages of the culture cycle, and is uniformly distributed below the liquid surface under various medium depth conditions. This feature makes the bioreactor system 1000 and scaffold assembly 200 suitable for culturing at 10%-100% capacity, compatible with a wide range of culture sizes and seed cell numbers. The multiple scaffold layers 220 in the scaffold assembly 200 may allow initially inoculated cells to be distributed across the layers of scaffold assembly 200, with a highmultiplicity of expansion in a single reactor, reducing the number of secondary cultures, and with lower labour costs and risk of bacterial contamination.

[0162] [Fig. 5] shows an example of a scaffold sheet 220 formed of Volvariella volvacea (straw mushroom) mycelial fibres in a dry form. The scaffold sheet 220 may therefore comprise or be formed from edible mycelial fibres. The scaffold sheet 220 may be a mycelial sheet comprising intertwined mycelial fibres and the mycelial scaffold sheet 220 is configured to support adhesion of one or more cells on one or more surfaces of the mycelial scaffold sheet 220. In some examples, the mycelial scaffold sheet 220 comprises ergothioneine. The ergothioneine may be from a fungal mycelium used to form the mycelial scaffold sheet 220. The mycelial scaffold sheet 220 may comprise one or more other natural compounds (e.g. amino acids, peptides, lipids, vitamins, etc.) from a fungal mycelium used to form the mycelial scaffold sheet 220. [Fig. 6] provides analytic data of a scaffold sheet 220 formed of Lentinula edodes (shiitake mushroom) mycelial fibres. This data shows the presence of polysaccharides, lipids, proteins, chitin, vitamins in the scaffold sheet 220. These assays indicate that the mycelium scaffold sheet 220 is nutritionally beneficial. Examples of the one or more natural compounds as provided in [Fig. 6] include but are not limited to methyl tridecanoate, azelaic acid dimethyl ester, methyl tetradecanoate, methyl pentadecanoate, methyl palmitate, methyl linoleate, methyl a-linoleate, methyl stearate, thiamine (B1), riboflavin (B2), nicotinic acid (B3), niacinamide (B3), pantothenic acid (B5), biotin (B7) and choline. The one or more other natural compounds may have benefits, for example improving cell adhesion and cell proliferation during cell cultivation. It will be appreciated that scaffold sheets formed of mycelial fibres from fungal species other than Lentinula edodes (shiitake mushroom) may have one or more similar natural compounds as described above with reference to [Fig. 6] albeit in different concentrations. The ergothioneine (or one or more other natural compounds) may be maintained in the mycelial scaffold sheet 220 during manufacture of the mycelial scaffold sheet 220 because the mycelial scaffold sheet 220 may be formed by a manufacturing method that does not involve a chemical process which destroys or removes ergothioneine (or one or more other natural compounds). In some examples, the manufacturing method 220 involve wholly mechanical processes. In some examples, the manufacturing method comprises a method according to an aspect of the present disclosure.

[0163] Ergothioneine is a rare amino acid with strong antioxidant capacity that can only be synthesized in some bacteria and fungi, whereas animals and plants cannot synthesize ergothioneine on their own but can absorb it and accumulate it in their bodies (Fujitani Y et al., 2018; Cheah IK et al., 2012). Reports have shown that ergothioneine reduces the extent of hydrogen peroxide-induced DNA damage (Colognato R et al., 2006), inhibits peroxy nitrite- induced oxidative DNA damage (Aruoma Ol et al., 1999), and has a protective effect against palmitic acid induced cell death (Laurenza I et al., 2008). An ergothioneine-containing mycelialscaffold sheet 220 enhances cellular resistance to oxidative stress, thereby improving the success of cell culture of the mycelial scaffold sheet 220, for example in the cultivation of food products.

[0164] In some examples, the mycelial scaffold sheet 220 comprises ergothioneine at a concentration of about 0.1 mg / g to about 15.0 mg / g of the dry mycelial scaffold sheet 220. In some examples, the mycelial scaffold sheet 220 comprises ergothioneine at a concentration of about 2 mg / g to about 12.0 mg / g of the dry mycelial scaffold sheet 220.

[0165] The mycelial scaffold sheet 220, particularly one formed by a process described in the present disclosure, comprises a multilayered network structure formed by intertwined mycelial fibres with an average diameter of about 1 pm to about 10 pm. The mycelial scaffold sheet 220 comprises a non-woven sheet. A non-woven sheet comprises fibres which are arranged randomly unlike a woven sheet which comprises fibres arranged in an organised manner. In some examples, the non-woven mycelial scaffold sheet 220 comprises randomly arranged mycelial fibres. A non-woven sheet may be isotropic whereas a woven sheet may be anisotropic. For example, a woven sheet may exhibit a first extensibility in a first direction (e.g. along a warp direction) and a second extensibility in a second direction (e.g. along a weft direction), where the first extensibility is different from the second extensibility. In a non-woven sheet, the extensibility of the sheet may be substantially the same in all directions. In some examples, the mycelial fibres comprise comminuted fibres from a fungal mycelium.

[0166] The mycelial scaffold sheet 220 may have semi-permeable membrane properties. Experiments have demonstrated that mycelium scaffold sheets 220 prepared by a process described in the present disclosure is permeable to small molecules (e.g., water molecules, glucose molecules, amino acids, etc.). The permeability to large molecules (e.g., proteins, polysaccharides) depends on the mycelium scaffold making process. For example, when using Volvariella volvacea (straw mushroom) mycelium and mushroom polysaccharide as raw materials and controlling the thickness of mycelium slurry at 2mm, the resulting scaffold sheet 220 can be permeable to substances with molecular weights of up to 1 ,000 Daltons. When shiitake (Lentinula edodes) mycelium and mushroom polysaccharide are used as raw materials, and the thickness of the spreading of the mycelium slurry is controlled to be 0.5 mm, the resulting scaffold sheet 220 can approximately pass through a substance with a molecular weight not exceeding 100,000 Daltons. Therefore, the permeability of the scaffold sheet to molecular weights, for example between 1,000 and 100,000 Daltons may be controlled by controlling the thickness and pore size of the mycelium scaffold sheet 220.

[0167] Thickness of the mycelial scaffold sheet 220 depends on its use. In some examples, the mycelial scaffold sheet 220 has a thickness of about 10 pm to about 100 pm. In some examples, the scaffold sheet 220 may comprise varying thicknesses along its width and / or length, for example, the scaffold sheet 220 may have a thicker central portion 222 relative to its end portions 223 to allow robust connection with the one or more connector strips 230.

[0168] While the scaffold sheet 220 is shown to have a substantially square shape, it will be appreciated that the scaffold sheet 220 may be formed into other shapes which include but are not limited to a rhombus, rectangle, triangle, circle, oval, pentagon, hexagon, heptagon and octagon.

[0169] The mycelial scaffold sheet 220 may be highly resistant to liquid culture environments, and the scaffold sheet 220 can be continuously oscillated in a culture medium solution for 3 months or more without distortion or loosening of the original shape. This feature makes the mycelium scaffold 210 of the present disclosure strong enough to be used in cell cultures of various growth cycles.

[0170] At the same time, the mycelium scaffold 210 and scaffold sheets 220 are able to be chewed and broken like food. This gives it characteristics that make it edible unlike ordinary paper or cotton. The mycelial scaffold sheet 220 of the present disclosure may be adjusted for edibility and palatability, at 1 / 1 Oth natural bite. The upper limit of scaffold strength may be developed based on food palatability and the lower limit of scaffold strength may be chosen based on the forces applied in the culture environment. In an example, the scaffold sheet 220 of the present disclosure has good stability and moderate strength, which makes the scaffold sheet 220 less prone to breakage or fragmentation during incubation and cell cultivation, and suitable for chewing and swallowing as a food product or part thereof. The properties of the scaffold sheet 220 as discussed below allow the scaffold sheet 220 to be sufficiently robust and resistant to damage when exposed to cell culture and incubation conditions (e.g. agitation through aeration), but insufficient to burden or harm a consumer with significant chewing and swallowing (e.g. masticatory muscle myalgia).

[0171] In a test of scaffold sheets 220 formed from Volvariella volvacea (straw mushroom) mycelium, a single scaffold strip (wet, flat), referred to as a reference portion in the present disclosure, of 500mm2(10mm width) can withstand a static tensile force between 0.049N and 0.147N, with most of the samples withstanding 0.098N and fracture occurring after a dynamic tensile force of 0.1N. This allows for final edible products (e.g. 5cm wide, containing 20 layers of cell sheet, non-fried) products to fracture at a force of 10N (about 1 kg), meaning that excessivebite forces are not required (natural teeth have a bite force of around 40kg (-400N), artificial teeth usually have a bite force greater than 10kg (-100N)).

[0172] Maximum velocity water flow tests in a simulated bioreactor system showed full structural integrity and zero breakage or fragmentation of 12 mm wide by 50 mm long mycelium scaffold sheets 220 according to an embodiment of the present disclosure after 48 hr of agitation in liquid stirred at 600 rpm without direct contact with the stirrer. The 12 mm wide, 50 mm long mycelium scaffold sheets 220 were also agitated in liquid for 48 hrs in an air lift reactor with an air flow rate > 10 L / min with no breakage or fragmentation.

[0173] As shown in [Fig. 7], mycelium scaffold sheets 220 formed from Volvariella volvacea (straw mushroom) mycelium showed a decrease in strength following submersion under alkaline conditions and strong oxidising conditions, with Fracture Stress reduced to about 50% of the control. There was also some reduction in strength in acidic conditions and high sugar and salt environments. These test conditions were far harsher than widely used cell culture environments, suggesting that the mycelium scaffold sheets 220 can cope with standard animal cell culture environments (e.g. pH 6.8-7.6, no more than 1% glucose and no strong oxidants).

[0174] In some examples, the mycelial scaffold sheet 220 is extensible between about 5% to about 25% of its unextended length in response to a force applied to the mycelial scaffold sheet 220. In some examples, the mycelial scaffold sheet 220 is extensible between about 7% to about 21% of its unextended length in response to a force applied to the mycelial scaffold sheet 220. In some examples, the mycelial scaffold sheet 220 comprises a Young’s Modulus of between about 3.0 MPa to about 7.0 MPa. In some examples, the mycelial scaffold sheet comprises a Young’s Modulus of between about 3.5 MPa to about 6.5 MPa. In some examples, the mycelial scaffold sheet comprises a tensile strength of between about 0.1 MPa to about 0.6 MPa.

[0175] In an example provided by [Fig. 8], tensile tests show that the elongation at break of mycelium scaffold sheet samples formed from Volvariella volvacea (straw mushroom) mycelium can range from about 7% to about 21% ([Table 1)). Further as shown in [Table 1], the mycelium scaffold sheet samples exhibit a Young’s Modulus of about 3.98MPa to about 6.15Mpa, and a tensile strength of about 0.1 MPa to about 0.6 MPa.

[0176] [Table 1]: Calculated mechanical properties of mycelium scaffold sheet samples formed from Volvariella volvacea (straw mushroom) mycelium.Sample Stress at break Elongation at break ModulusMPa % MPa1 0.13718 20.646 3.983742 0.32486 7.764 4.791293 0.53838 13.196 4.804834 0.51468 12.769 4.816645 0.40689 9.064 6.15225

[0177] Another tensile strength experiment was conducted on hydrated samples of Volvariella volvacea (straw mushroom) mycelium sheets of weight 0.01g, thickness <0.1 mm and dimensions 12x30mm. The samples exhibited a Young’s modulus of 4.9 ± 0.8 MPa, with elongation at break of 13 ± 5 % and a tensile strength of 0.4 ± 0.2 MPa.

[0178] Stirred tank bioreactor simulations were also carried out with Volvariella volvacea (straw mushroom) mycelium scaffold sheet samples less than 0.1mm in thickness, submerged at a water depth of 200mm (1 ,96kPa), with one piece centrally mounted of dimensions radius 30mm by diameter 60mm for bilateral shear stress and agitation, and another piece mounted to the wall of the bioreactor (W)20mm by (L)50mm for lateral shear stress and agitation. The experiment showed an exemplary scaffold sheet of the present disclosure can withstand high rotary speeds of 600rpm for 48 hours with zero structural breakage or fragmentation. To further test durability, the samples were also subjected to periodic stirring each time for 90 minutes at 800rpm, which is more than 5 times the maximum speed commonly used in animal cell culture. Full structural integrity remained even at 1000rpm.

[0179] Gas-lift bioreactor simulations were also carried out with the same material and conditions. Scaffold sheet pieces were mounted above the micro-porous air diffuser / sparger to allow contact with air bubbles, and to simulate the agitation and turbulence caused by the rising bubbles. The experiment showed an exemplary scaffold sheet of the present disclosure retains full structural integrity at exceedingly high rates of gas flow of more than 10L / min, which is more than 10 times the maximum air flow normally used in cell culture, which is typically 0.5L to 1 .0L / min (VVM >0.01 to <0. 1). For bioreactors with additional media circulation system, mycelium scaffold sheets of the present disclosure can tolerate media flow rates of up to 116 L / min (equivalent to a linear velocity of 1.08 m / s), based on the shear force generated by the horizontal flow of media between the layers of a large-area (0.9m*0.9m) scaffold. This means that the lower limit of scaffold sheet tolerance is still 3-4 times higher than conventional media flow rate.

[0180] The mycelial scaffold sheet 220 formed of edible mycelial fibres may be used in the cultivation of edible food products. In particular, the edible mycelial fibres allow for the harvesting of food-grade products without the need for additional harvest and post-harvest processing steps and costs. The edible scaffold sheet 220 therefore does not require enzymatic digestion toseparate the cells for example from a plastic scaffold during harvest, making it easy to handle and with less risk of foreign material residues (e.g. plastic fragments or microplastics). Unlike plastic scaffolds, there is no need to trypsin-wash the cells off the scaffold sheets 220 at the end of the culture, but that the scaffold sheets 220 of the present disclosure are harvested and eaten together with the cultivated food product. Since the entire three-dimensional structure of the scaffold assembly 200 (excluding the float) is made up of mycelium, it is unlike plastic scaffold, which is considered a foreign substance if it is mixed with the food product.

[0181] In addition, the mycelial scaffold structure 210 and scaffold sheet 220 can provide a cultivated meat product with a texture similar to that of meat fibres, compensating for the lack of chewiness that is usually associated with the loose structure of cellular meat, improving the organoleptic properties of the cultivated meat product. The mycelial scaffold structure 220 may therefore be edible and comprises an elasticity configured to provide a sensation of chewiness.

[0182] One of the substances which may be necessary for the synthesis of proteoglycans in the cartilage matrix of human joints is glucosamine (2-Amino-2-deoxy-p-D-glucose), a component that is usually present in the form of an N-acetyl derivative. A scaffold sheet 220 formed from mushrooms and mushroom mycelium has natural chitin which is a polymer of acetylaminoglucose (p-(1 ,4)-2-acetamido-2-deoxy-D-glucose), and this scaffold sheet 220 provides the cells that are being cultivated with a physiological environment similar to that in the vicinity of the cartilage tissues, which is more conducive to normal cell growth. A mycelial scaffold sheet 220 formed based a manufacturing method of the present disclosure substantially retains the natural chitin of the mycelium it is formed from, hence supplemental chitin need not be added during the manufacturing process or a minimal amount of supplemental chitin may only be needed. Additionally or alternatively, the mycelial scaffold sheet 220 preserves the natural conformation, topological structure, molecular packing, and supramolecular architecture of chitin in mushrooms, which differs from the structure of reconstituted chitin scaffolds. This also contributes to a texture that closely resembles that of natural mushrooms.

[0183] Many prior art scaffolds still use animal derived materials (e.g. bones, skins), and fail to completely depart from the harvesting or farming of natural animals, resulting in ethical and religious debate. In contrast, a scaffold of the present disclosure comprises material exclusively from plants and / or fungi, which may be screened through bioinformatics analysis and extensive experimental testing to help provide the cells with an environment close to that of an animal's body. In some examples, the scaffold of the present disclosure comprises scaffold 210 which comprises mycelial scaffold sheets 220 which comprise material exclusive from fungi. Therefore, a scaffold or a scaffold sheet of the present disclosure (e.g. scaffold sheet 220) may be devoid of material derived from animals. This can be a truly a ‘cruelty free’ and ‘vegetarian’ ingredient thatdoes not harm or use animals products, and a process that brings clarity to the cultured meat product concept. The mycelial fibres used in the formation of the scaffold sheet 220 may be from one or more of fungal species comprising: Volvariella volvacea (Straw Mushroom), Lentinula edodes (shiitake mushroom), Hericium erinaceus (lion's mane mushroom), Ganoderma lucidum (lingzhi mushroom I reishi mushroom), Pleurotus eryngii (king oyster mushroom), Pleurotus ostreatus (oyster mushroom), Flammulina velutipes (enoki mushroom), Agaricus bisporus (button mushroom), Grifola frondosa (maitake mushroom) and Rhizopus oligosporus. It will be appreciated that the mycelial fibres used in the formation of the scaffold sheet 220 are not limited to the fungal species to which tests and experiments have been conducted on and as disclosed in the present disclosure, and that mycelial fibres from other fungal species may be utilized to form a scaffold sheet of the present disclosure depending on its use and application. For example, a fungal species (e.g. Grifola frondosa) may be chosen based on a taste profile it may impart to a consumer when the scaffold sheet, formed of mycelial fibres of such species, is used in the cultivation of a meat product. In some examples, the mycelial fibres are from a plurality of fungal species, for example, the mycelial scaffold sheet 220 may be formed of mycelial fibres from the species Lentinula edodes (shiitake mushroom) and Hericium erinaceus (lion's mane mushroom). In another example, the mycelial scaffold sheet 220 may be formed of mycelial fibres from the species Lentinula edodes (shiitake mushroom) and Volvariella volvacea (Straw Mushroom).

[0184] In addition to chitin, the scaffold sheet 220 may contain mushroom polysaccharides and polypeptides. In an example, the polysaccharides range from about 35% to about 80% of the dry weight of the scaffold sheet 220. In an example, the polypeptides range from about 10% to about 30% of the dry weight of the scaffold sheet 220. The polysaccharides and polypeptides (and their respective amounts relative to the dry weight of the scaffold sheet) can provide a suitable environment for cell culture.

[0185] To further enhance adsorption of animal cells to the scaffold sheet 220, the scaffold sheet 220 may comprise one or more additive compounds. The additive compounds may be positively charged when the scaffold sheet 220 is in use, i.e. in culture medium. The additive compounds can be edible mushroom proteins (or plant proteins), peptides or amino acid mixtures obtained by enzymatic processing of plant proteins, lysine or polylysine, and chitosan. In an example, the additive compounds are selected from a group comprising lysine and chitosan. The lysine may be a polylysine. Mycelial scaffolds and scaffold sheets with added chitosan not only accelerate the attachment of animal cells after inoculation, but also inhibits the growth of undesirable microorganisms, has some ability to avoid or prevent microbial contamination, and reduces the risk of bacterial contamination during cell culture.

[0186] Scaffold sheet samples formed either of Lentinula edodes (Shiitake mushroom) or Volvariella volvacea (straw mushroom) mycelial fibres, were tested using a fish myogenic cell, and most cells affixed to the scaffold sheet samples within 24 hours, and the cells grew in a similar cellular state when compared to control cultures on cell culture plates. Scanning electron microscopy and fluorescence staining microscopy showed that the cell morphology and growth status were normal. The samples were in 0.9% NaCI submerged for more than 90 days, yet retaining its structural integrity in transport, growth experiments, and subsequent harvesting and handling for imaging purposes.

[0187] Method of Forming a Scaffold Sheet

[0188] According to an aspect of the present disclosure, there is a method of forming a biocompatible scaffold sheet (e.g. mycelial scaffold sheet 220), the method comprises the step of: comminuting plant and / or fungal material; forming a suspension in a liquid form from the comminuted plant and / or fungal material; spreading the suspension out on a support; and micro piercing the spread suspension to cause fibres in the plant and / or fungal material to intertwine.

[0189] The step of comminuting plant and / or fungal material comprises reducing the size of the plant and / or fungal material from a larger average particle size to a smaller average particle size, and includes but is not limited to crushing (e.g. roller crushing, high pressure crushing, mortar and pestle, food processor grinding), grinding, cutting, vibrating, pulverizing, ultrasonic disintegration, shredding, milling (e.g. cryogenic milling), electrical discharge, attrition, and other mechanical or physical methods of processing. In an example, the step of comminuting plant and / or fungal material excludes any chemical processes that chemically breaks down the plant and / or fungal material. The step of comminuting damages the mushroom cell to remove the soluble components, reducing the content of potentially cytotoxic components in the product (e.g., glycosides and phenolics), while retaining the chitin and desirable proteins, e.g. membrane protein, so that the resultant scaffold and scaffold sheet remain cell-absorbent. In an example, the method substantially reduces the content of small molecules such as phenols and organic acids in order to avoid stressing the cells or affecting the ionic environment. The method therefore causes fibre dispersion and reconstruction of the mycelium.

[0190] To enhance the adsorption of cell to the scaffold sheet, additional positively charged components (i.e. additive compounds) may be added to the suspension solution. These components may be edible mushroom proteins (or plant proteins), peptides or amino acid mixtures obtained by enzymatic processing of plant proteins, lysine or polylysine, and chitosan. Mycelial scaffold sheets with added chitosan not only accelerates the attachment of animal cells after inoculation, but also inhibits the growth of undesirable microorganisms, has some ability to avoid or prevent microbial contamination, and reduces the risk of bacterial contamination during cell culture.

[0191] In an example, the method comprises processing the mycelium of a fungi into mycelial fibres with high dispersion by physical crushing (e.g. grinding), mixing the crushed mycelium with polysaccharides (e.g. edible polysaccharides such as pea starch), fungal and / or plant proteins (e.g. Ganoderma lucidum proteins, mushroom proteins, soy proteins, etc.) and a liquid (e.g. water) to form a mycelial fibres suspension, spreading the mycelial fibres suspension on a tray or molding belt with a fine mesh at the bottom at 0.5-2mm thickness when it is wet, and forming the scaffold sheet by micro-piercing (e.g. via a micro-needle piercing or micro-waterjet piercing method) to cause the mycelial fibres to intertwine (similar to the non-woven blown fibre method) with each other. The shape of the scaffold sheet may depend on the shape of the mould the mycelial fibres suspension is spread on. This intertwined suspension may then be compressed to dewater and then dried at certain temperatures for several hours, embodiments of drying may include air drying and contact drying. The dried product is then removed from the drying device as a sheet. This edible scaffold is biocompatible with animal cell cultivation. Industrial equipment such as fibre shears (capacity 500-800kg / h), small reactors (capacity 1000-2000L), rotary drum dryer (drying capacity 40-400kg / h), etc. can be used to form a scaffold sheet of the present disclosure.

[0192] After the scaffold sheet is formed, two or more of the scaffold sheets may be connected together to form a scaffold structure by interweaving the sheets with one or more connector strips of plant and / or fungal material as shown in [Fig. 2] and [Fig. 3]. The step of connecting the scaffold sheets may comprise placing the scaffold sheets apart, e.g. at a predetermined distance, so as to create a space between the scaffold sheets to allow for culture media flow and animal cell growth, prior to interweaving the sheets with the one or more connector strips.

[0193] Bioreactor System

[0194] [Fig. 9] provides a schematic diagram of a bioreactor system 2000 according to an embodiment of the present disclosure and its components. The bioreactor system 2000 is similar to the bioreactor system 1000 except for certain system components. Unless described as beingdifferent below, the features, functionality, alternatives and uses of bioreactor system 2000 are as described for bioreactor system 1000, or any other bioreactor system described in the present disclosure. The bioreactor system 2000 may be used for an adherent cell culture. The bioreactor system 2000 may be used in the culture of cultivated food products, and may be suitable not only for adhesion-dependent cell types (e.g. myoblasts, adipocytes), but also for culturing partially adherent or suspension cell types (e g. fibroblasts, lymphocytes).

[0195] The components of the bioreactor system 2000 include a cell culture vessel 2100, a gas exchange vessel 2400 (also referred to as a gas exchanger unit 2400 in the present disclosure), one or more compression reflux liquid pumps 2300 (and / or compression liquid pumps 2300’), a metabolite separation device 2800, an optional culture medium incubation unit 2900 (an example shown in [Fig. 14] but not shown in [Fig. 9]) and one or more conduits 2600 connecting the various components of the bioreactor system 2000. Components of bioreactor system 2000 may be collectively referred to as bioreactor system components in the present disclosure. The bioreactor system 2000 further comprises a scaffold assembly 2200 housed in cell culture vessel 2100. The bioreactor system 2000 may include an optional sterilization jacket (an example is shown in [Fig. 13]). The bioreactor system 2000 may comprise one or more auxiliary parts, identical or similar to like parts of bioreactor system 1000, for example, sensors (e.g. oxygen (O2) sensor2101, carbon dioxide (CO2) sensor 2103, pH sensor 2102, and temperature sensor 2104), auxiliary pumps and control devices (e.g. temperature control module 2500). The bioreactor system 2000 may comprise one or more control devices to control one or more components of the bioreactor system 2000.

[0196] The cell culture vessel 2100 can be made of glass, stainless steel with or without a window, or plastics that are food-safe and resistant to high-temperatures during sterilization or retort. The cell culture vessel 2100 comprises a cover 2105 with ports. The cover 2105 may be a removable cover. The ports may be configured to position one or more components such as sensors and conduits (for transport of culture medium). The cell culture vessel 2100 comprises a first outlet 2107a. The first outlet 2107a is positioned on the cover 2105 and may be provided in the middle of the cover 2105. The first outlet 2107a may be an upper outlet 2107a. The cell culture vessel 2100 also comprises a second outlet 2107b provided in a base portion of one side of the vessel 2100. The second outlet 2107b may be a lower outlet 2107b. The first outlet 2107a is positioned further, relative to the second outlet 2107b, from a surface upon which the cell culture vessel 2100 is supported. The cell culture vessel 2100 comprises an inlet 2108 in a base portion of one side of the vessel 2100. The second outlet 2107b and inlet 2108 may be positioned proximal one another. It will be appreciated that the outlets 2107a, 2107b and inlet 2108 may be arranged differently on the cell culture vessel 2100, for example, both first and second outlets 2107a, 2107b may be arranged on the cover 2105, or both first and second outlets 2107a, 2107bmay be arranged on the side of the cell culture vessel 2100. In an example, the first and second outlets 2107a, 2107b are distanced from each other. In some examples, there is more than one inlet 2108 and / or there is more than two outlets 2107a, 2107b. In some examples, the inlet 2108 may be positioned at a distance from one or both outlets 2107a, 2107b, such that culture medium flowing into the cell culture vessel 2100 via the inlet 2108 may flow through a substantial portion of the cell culture vessel 2110 before exiting one or both outlets 2107a, 2107b. In some examples, one or both outlets 2107a, 2107b and / or the inlet 2108 may be provided with a conduit that extends into the cell culture vessel 2105. In some examples, the lengths of the conduits that extend into the cell culture vessel 2100 from the first and second outlets 2107a, 2107b and / or inlet 2108 may be the same or different. For example, if both first and second outlets 2107a, 2107b are provided on the cover 2105, the conduit extending from second outlet 2107b may extend closer to the base of the cell culture vessel 2100 relative to the conduit extending from first outlet 2107a. This allows different flow paths (described below) of the bioreactor system 2000 to be utilized during different phases of cell culture. The cover 2105 or the cell culture vessel 2100 can be fitted with a variety of commercially available sensors (e.g. pH sensor 2102, temperature sensor 2104, dissolved oxygen detector 2101 , CO2 sensor 2103 etc.) to monitor one or more parameters of the cell culture in the cell culture vessel 2100, for example to monitor cell growth. The cell culture vessel 2100 may have an inoculation port (not shown) and a feeder port 2109 for adding culture medium. The cell culture vessel 2100 can be insulated on its outside, but the insulation may need to include a transparent window section for inspection during the cell cultivation process.

[0197] Unlike the bioreactor system 1000, the bioreactor system 2000 comprises two flow paths A, B along which culture medium can flow, and these flow paths will be described with cell culture vessel 2100 as a reference. In flow path A, efferent culture medium is pumped out of the upper part of the cell culture vessel 2100 via first outlet 2107a by a compression reflux liquid pump 2300a through a conduit 2600 into the metabolite exchange device 2800. Culture medium exiting the metabolite exchange device 2800 thereafter enters the gas exchanger unit 2400, and oxygenrich culture medium is returned to the cell culture vessel 2100 via inlet 2108 as afferent culture medium at the lower part of the vessel 2100. In flow path B, efferent culture medium is pumped out of a lower part of the cell culture vessel 2100 via second outlet 2107b by a compression reflux liquid pump 2300b and into the gas exchange vessel 2400, while oxygen-rich afferent culture medium is returned to the cell culture vessel 2100 via inlet 2108 at the lower part of the cell culture vessel 2100. In flow path A, culture medium is pumped indirectly to the gas exchanger unit 2400, i.e. the culture medium has to first pass through the metabolite exchange device 2800, and in flow path B, culture medium is pumped directly to the gas exchanger unit 2400. Check valves are positioned along the flow paths, e.g. in conduit 2600 to direct flow of the culture medium along anintended flow path. For example, check valve 2303 prevents culture medium from bypassing gas exchange vessel 2400 and entering directly into cell culture vessel 2100 via compression reflux liquid pump 2300b, or minimizes flow of culture medium from entering into cell culture 2100 via compression reflux liquid pump 2300b. In addition, check valve 2303 prevents culture medium from gas exchange vessel 2400 from back-flowing into compression reflux liquid pump 2300b or back-flowing into cell culture vessel 2100.

[0198] Activation of flow path A and / or flow path B depends on the volume of culture liquid in the cell culture vessel 2100. For example, when the volume of the culture medium in the cell culture vessel is above a predetermined volume (e.g. cell growth stage), flow path A may be activated to cause the culture medium in the cell culture vessel 2100 to be pumped out of first outlet 2107a to the gas exchanger unit 2400. In some examples when the volume of the culture medium in the cell culture vessel is above a predetermined volume (e.g. cell growth stage), both flow paths A and B may be activated to cause the culture medium in the cell culture vessel 2100 to be pumped out of first and second outlets 2107a, 2107b, to the gas exchanger unit 2400. Activation of both flow paths A and B may increase oxygenation in the cell culture vessel 2100. When the volume of the culture medium in the cell culture vessel is below a predetermined volume (e g. cell inoculation stage), only flow path B may be activated to cause the culture medium in the cell culture vessel 2100 to be pumped out of only the second outlet 2107b, to the gas exchanger unit 2400.

[0199] Bioreactor system 2000 comprises compression reflux liquid pumps 2300a, 2300b and a compression liquid pump 2300’. A compression liquid pump 2300’ is similar to a compression reflux liquid pump 2300 as described below, except that the compression liquid pump 2300’ does not comprise a return branch 2306. In some examples, the compression liquid pump 2300’ comprises a compression reflux liquid pump, a peristaltic pump or a diaphragm pump. In some examples, one or more of the compression reflux liquid pump 2300a, 2300b, comprises a compression liquid pump 2300’, a peristaltic pump or a diaphragm pump.

[0200] One or more filters 2106 may be provided to the cell culture vessel 2100, e.g. at an end of a conduit 2600 to prevent or minimize cells in the cell culture vessel 2100 from flowing out of the cell culture vessel 2100 when culture medium is pumped out of the cell culture vessel 2100.

[0201] The cell culture vessel 2100 includes a scaffold assembly 2200 to support adhesion of cells. The scaffold assembly 2200 may be a scaffold assembly according to an embodiment of the present disclosure, for example scaffold assembly 200. The scaffold assembly 2200 may be formed of a biocompatible material. The biocompatible material may be an edible material, for example a material derived from plants and / or fungi. The scaffold assembly 2200 may comprisemulti-layered scaffold structure for example multiple scaffold sheets that increase the surface area for adhesion and exposure of the adhered cells to the cultivation medium in the cell culture vessel. One of more scaffold sheets may be a scaffold sheet according to an embodiment of the present disclosure, for example mycelial scaffold sheet 220. The scaffold assembly 2200 may be provided in a fixed or unfixed position in the cell culture vessel 2100. The scaffold assembly 2200 may be connected, for example removably connected to a wall of the cell culture vessel 2100.

[0202] Compression Reflux Liquid Pump

[0203] As illustrated in [Fig. 10], the compression reflux liquid pump 2300 comprises a controller (not shown), a rigid or laminate plate 2301 , and a soft textured I flexible, fully enclosed spherical or ellipsoidal cavity (balloon) 2302, i.e. compressible pump chamber 2302). The balloon 2302 may be made of silicone, rubber, or other food-safe contact materials resistant to sterilizing treatment of at least about 121 °C.

[0204] Culture medium is received by a pump chamber inlet 2304 of the compression reflux liquid pump 2300 from a component upstream of the compression reflux liquid pump 2300. The inlet 2304 comprises a check valve 2303. Culture medium from upstream of the compression reflux liquid pump 2300 enters the balloon 2302 via the chamber inlet 2304. Culture medium may be conveyed to and from the compression reflux liquid pump 2300 via one or more conduits 2600. Culture medium exits a pump chamber outlet 2305 to a component downstream the compression reflux liquid pump 2300. The outlet 2305 comprises a check valve 2303. The check valves 2303 prevent or minimise reverse flow of the culture medium.

[0205] An exterior of the ballon 2302 is in contact with plates 2301. In some examples, the compression reflux liquid pump 2300 may comprise two or more plates 2301 , e.g. two, three, four, five or six plates. The plates 2301 are configured to be actuated towards each other to compress the balloon 2302 that is positioned between them. When plates 2301 are actuated towards each other, the balloon 2302 becomes compressed (i.e. systole) to a constricted configuration and when the plates 2301 are actuated away from each other, the balloon 2302 is allowed to expand (i.e. diastole) to a dilated configuration. In some examples, the exterior surface of the balloon 2302 is attached to the plates 2301 and movement of the plates 2301 away from each other pulls apart the balloon 2302, thereby causing it to expand. Movement of the plates 2301 towards and away from each other is under the control of the controller. Actuation of the plates 2301 towards or away from each other may be by motor actuation and / or magnetic induction. In some examples, the plates 2301 may not be actively actuated away from each other but a force used to actuate the plates 2301 towards each other may be removed and the plates 2301 are allowed to passively move away from each other under the expansion of the balloon 2302. One or more surfaces ofthe plates 2301 may be flat (i.e. planar), curved and / or comprise a shape that is suitable for its application. In some examples, one or more plates 2301 comprises a flat (i.e. planar) plate, a curved plate or has a shape suitable for its application. When the balloon 2302 is diastolic (i.e. from the constricted configuration to the dilated configuration), the internal pressure of the balloon 2302 decreases and the culture medium is drawn into the balloon 2302 through the inlet 2304. When the balloon is systolic (i.e. from the dilated configuration to the constricted configuration), the internal pressure of the balloon 2302 increases and the culture medium is driven out of the outlet 2305.

[0206] In some examples, the outlet 2305 is split into two fluid channels, a first fluid channel providing a first portion of the culture medium flowing out of the pump 2300 to a component (e.g. gas exchanger unit 2400) downstream of the compression reflux liquid pump 2300, and a second fluid channel (i.e. return branch 2306) providing a second portion of the culture medium to a portion upstream of the compression reflux liquid pump 2300. The check valve 2303 may be positioned in the first fluid channel. A check valve may be absent in the return branch 2306. The return branch 2306 causes part of the outflow of culture medium from the compression reflux liquid pump 2300 to flow back upstream of the compression reflux liquid pump 2300. During systole of the balloon 2302, this can increase pressure upstream of the compression reflux liquid pump 2300 thereby causing a flushing of a portion upstream of the compression reflux liquid pump 2300. Due to the higher pressure in the balloon 2302 (i.e. during systole), culture medium upstream of the compression reflux liquid pump 2300 is prevented or minimized from entering the balloon 2302. A check valve 2303 in the inlet 2304 positioned downstream of the portion in which culture medium is returned upstream of the compression reflux liquid pump 2300 also prevents or minimises upstream culture medium from entering the balloon 2302 because during systole, the check valve 2303 will be in a closed configuration. The flushing of a portion or component upstream of the compression reflux liquid pump 2300 may be beneficial to dislodge or disperse material (e.g. cells) that may clog upstream of the compression reflux liquid pump 2300 thereby affecting flow of the culture medium within bioreactor system 2000. This may occur in arrangements where a filter is positioned upstream of the compression reflux liquid pump 2300. In some examples, the flow rate of the second portion of the culture medium in the second fluid channel is lower relative the flow rate of the first portion of the culture medium in the first fluid channel.

[0207] In an example with reference to compression reflux liquid pump 2300a of [Fig. 9], one end of the balloon 2302 is connected to a one-way valve 2303 (i.e. check valve) and a connector 2307, leading to the cell culture chamber 2100 below the liquid surface, and the front end of 2307 of which is a filter assembly 2106 (e.g. a layer of 0.5 pm ceramic microporous filters and a layer of stainless-steel filters with a pore size of 200 pm). The other end of the balloon 2302 isconnected to another one-way valve 2303 and a diverter, and the diverter is connected to two outlet pipes, one of which leads to the metabolite separation device 2800 and the gas exchanger unit 2400, and the other outlet pipe is connected to a pipe proximal to the microporous filter assembly 2106. The outer side of the balloon 2302 is attached to 2 rigid plates 2301, which are driven by a motorized controller to rhythmically move closer to each other and then further away from each other, resulting in repeated compression (systole) and diastole of the balloon 2302. The rigid plates 2301 each may comprise a surface that is proximal and faces the balloon during use. One or both of the surfaces of the rigid plates 2301 may be flat (i.e. planar), curved and / or comprise a shape that is suitable for its application. In some configurations, one or both of the rigid plates 2301 may be a flat (i.e. planar) plate, a curved plate or be shaped suitable for its application. When the balloon 2302 is diastolic, the internal pressure decreases and the culture medium is drawn into the balloon 2302 through the filter mesh and the microporous filter sheet; when the balloon is contracted (systole), the internal pressure increases and the liquid is squeezed out of the outlet section, part of the volume of liquid of which enters the metabolite separation device 2800 and part of which flows back to the upper side piping of the filter 2106. Due to the higher pressure inside the balloon 2302, the culture medium in the upper side of the filter 2106 cannot enter the balloon again due to pressure resistance, and these liquids can only return to the cell culture cavity 2100 through the microporous filters and stainless-steel filters in turn, thus achieving the effect of the reverse flushing filter.

[0208] Compared to a diaphragm pump, the compression reflux liquid pump 2300 (as well as compression liquid pump 2300’) has a closed chamber to avoid microbial contamination, and compared to a peristaltic pump, the cells are not harmed by being squeezed by the walls of the tubing because the walls of the channel are never completely fitted. In addition, the compression reflux liquid pump 2300 is provided with a return branch 2306, whereby a portion of the liquid pumped out each time is used to backwash the filter 2106, which prevents the filter 2106 from being clogged with cells or scaffold debris, as a self-cleaning function.

[0209] In the compression reflux liquid pump 2300 and the compression liquid pump 2300’, the liquid (i.e. culture medium) is isolated in a soft chamber 2302 integrated with the bioreactor (e g. the balloon 2302 is in closed fluid communication with the cell culture vessel 2100), avoiding contamination of the culture medium contacting the pump's diaphragm chamber 2302. These types of pumps may be configured to leave a distance between the walls of the chamber during compression (i.e. the balloon 2302 is not fully compressed), so even if a cell enters the compression chamber 2302, it is not physically crushed, avoiding cell damage caused by the liquid circulation process. The unique backwash design of the compression reflux liquid pump 2300 flushes cell or scaffold debris accumulated at the filter 2106 back into the bioreactor 2100,avoiding the constant accumulation of solids, preventing blockages, increasing the longevity of the unit, reducing maintenance operations, and enabling lowered cost of labour.

[0210] Another way to realize the function of backwashing the filter membrane is to forgo the liquid return branch pipe 2306, and change the inlet end 2304 check valve 2303 of the extrusion chamber 2302 of the compressed liquid pump into an partially closed 3-piece or 2-piece flap, so that when the volume of the compressed chamber 2302 is reduced, most of the liquid will flow out through the check valve 2303 at the outlet end 2305, and a small portion of the liquid will return through the partially closed flaps back to the inlet pipe 2304, so as to achieve the effect of rinsing the filter mesh 2106. While this design may be similar to the design of the compression liquid pump 2300’, the compression liquid pump 2300’ does not have a reverse flushing effect of its upstream component, i.e. reverse flow from the compression liquid pump 2300’ is absent or negligible to have a flushing effect.

[0211] The bioreactor system 2000 comprises 2 compression reflux liquid pumps; compression reflux liquid pump 2300b which is in fluid communication with the cell culture vessel 2100 via second outlet 2107b which is located below the side wall of the cell culture cavity (the height of which is about one-quarter to about one-third that of the cell culture vessel 2100) and is connected to the gas exchanger unit 2400, and compression reflux liquid pump 2300a which is in fluid communication with the cell culture vessel 2100 via first outlet 2107a located at the top of the cell culture vessel 2100 and is connected to the metabolite separation unit 2800.

[0212] Gas Exchange Vessel

[0213] The gas exchange vessel 2400 as shown in [Fig. 11] regulates a target gas species in the culture medium, for example enriching oxygen and regulating the carbon dioxide content of the cultivation media. The housing 2401 of the gas exchange vessel 2400 is a smaller container than the cell culture chamber 2100 and can be made of glass, ceramic, carbon fibre, stainless steel with or without a window, or food-safe and autoclavable plastics. In the centre of the gas exchange chamber there is a tube 2402 (i.e. first exchange chamber 2402), the sidewall (or wall as referred to in the present disclosure) of which is permeable to a target gas species and substantially impermeable to liquids. In some examples, the sidewall of the first exchange chamber 2402 comprises a hydrophobic semi-permeable membrane resistant to organic solvents. In some examples, the sidewall of the first exchange chamber 2402 may be of a folded, pleated, or ribbed design to increase surface area for gas exchange. The cavity 2403 (i.e. second exchange chamber 2403) between the outer wall 2401 of the gas exchange chamber 2400 and the centre pipe 2402 is filled with a target gas species liquid carrier 2403a comprising an amount of target gas species substantially different from an amount of target gas species in the inflowculture liquid to cause a target gas species concentration gradient between the inflow culture liquid and the target gas species liquid carrier 2403a, and the target gas species concentration gradient is configured to alter the amount of target gas species in the inflow culture liquid. The target gas species may be one or more of oxygen (O2), carbon dioxide (CO2), hydrogen (H2), and nitrogen (N2). The target gas species liquid carrier 2403a may be a synthetic oxygen carrier or bio-oxygen carrier (e.g., perfluorohexane, perfluorinated naphthalene, glutaraldehyde polymerized bovine hemoglobin aqueous solution, etc.). The bottom of the cavity 2403 may be provided with a second exchange chamber inlet to receive a gas comprising at least the target gas species for dissolution in the target gas species liquid carrier. The second exchange chamber inlet may be an air inlet 2404, an oxygen (O2) inlet 2405, and / or a carbon dioxide (CO2) inlet 2406. The second exchange chamber inlet may comprise gas inlets other than for air, O2 and CO2, such as a H2 and / or N2 gas inlet. The received gas is configured to moderate the concentration of gas species in the target gas species liquid carrier so as to generate and / or maintain the target gas species concentration gradient. In some examples, the second exchange chamber comprises a target gas species disperser to disperse the received target gas species and increase an interfacial area between the target gas species and the target gas species liquid carrier. For example, the target gas species dispersal comprises a bubbler.

[0214] The first exchange chamber 2402 comprises a first exchange chamber inlet 2402a. The first exchange chamber inlet 2402a is positioned at a first end portion of the gas exchanger unit 2400, and the second exchange chamber inlet 2404, 2405, 2406 is positioned at a second end portion of the gas exchanger unit, wherein the first and second end portions are opposing end portions of the gas exchanger unit 2400. In some examples, the second exchange chamber inlet 2404, 2405, 2406 is positioned closer to a ground surface relative to the first exchange chamber inlet 2402a so that a gas introduced to the target gas species liquid carrier 2403a, is able to pass through a substantial portion of the target gas species liquid carrier 2403a to increase residence time of the gas in the target gas species liquid carrier 2403a thereby improving dissolution of the target gas species (in the introduced gas) in the target gas species liquid carrier 2403a . The first exchange chamber 2402 comprises a first exchange chamber outlet 2402b which is positioned at the second end portion, proximal to the second exchange chamber inlet.

[0215] In some examples when in use and with reference to bioreactor system 2000 in [Fig. 9], the culture medium (i.e. inflow culture liquid) is filtered through a screen to remove any cultivated cells and then enters the intermediate pipeline 2402 of the gas exchanger unit 2400 through compression reflux liquid pump 2300b. During the flow process, the culture medium exchanges gases (including oxygen and carbon dioxide) with the target gas species liquid carrier 2403a on the other side of the semi-permeable membrane of the first exchange chamber 2402, and then returns to the cell culture vessel 2100 through the compression liquid pump 2300’ andthe temperature control unit 2500. When the bioreactor system 2000 is being used for adherent cell culture, compression liquid pump 2300’ may be a compression reflux liquid pump as described in the present disclosure, or it may be an ordinary peristaltic pump. When the bioreactor system 2000 is being used for suspension cell culture, compression liquid pump 2300’ may be a compression reflux liquid pump as described in the present disclosure.

[0216] Unlike known bioreactor systems which rely on passive gases dissolution and increasing the partial pressure of gases and / or increasing the surface area of dissolved gases by means of an aeration device, the bioreactor system 2000 of [Fig. 9] uses an indirect approach to oxygen dissolution, whereby the culture medium pass through a channel made of a microporous material (or a semi-permeable membrane) which is submerged in an exchange cell filled with a target gas species liquid carrier 2403a, such as a bio-oxygen-carrying agent (e.g., Perfluorohexane), with oxygen diffusing into the culture medium from the side of the target gas species liquid carrier 2403a. In the case of the second exchange chamber 2403 comprising Perfluorohexane, for example, the solubility of oxygen in Perfluorohexane is several hundred times higher than in water, so the target gas species liquid carrier 2403a can be enriched with oxygen by means of microporous aeration or by placing Venturi holes at the end of the second exchange chamber inlet 2404, 2405, 2406.

[0217] The gas exchange vessel 2400 greatly improves the rate of oxygen dissolution by using a target gas species liquid carrier 2403a such as a biological oxygen-carrying agent (e.g. Perfluorohexane) as an oxygen dissolving medium. At room temperature, the solubility of oxygen in water is only 9mg / L (i.e. 0.009g / L), and the dissolution rate is usually only 400-650mg / (m2*h) under high flow rate conditions, which means that the direct dissolution of oxygen into the water requires a large area of air-liquid surface. The efficiency and oxygen carrying capacity of biooxygen carriers in combination with oxygen is much higher than that of water, and the dissolved oxygen capacity can be as high as 8g / L. Therefore, it can rapidly absorb oxygen from air or oxygen-enriched air. Oxygen from the gas interface to the liquid interface has a high resistance, therefore the high oxygen-carrying capacity of the target gas species liquid carrier 2403a breaks through the bottleneck of gas-liquid interface mass transfer efficiency, then the oxygen only needs to complete its transfer between the two liquid interfaces by diffusion, improving the efficiency of gas exchange.

[0218] The gas exchange vessel 2400 also has good safety, the bio-oxygen-carrying agent itself (e.g. Perfluorohexane) has good biocompatibility, and has been used as a temporary substitute for oxygen-carrying blood in medicine. In addition, this device 2400 is to export the cell- free medium separately, and exchange gas with the bio-oxygen-carrying agent isolated on the other side of the microporous membrane, which provides double isolation, and the cells do notcome into direct contact with the bio-oxygen-carrying agent 2403a, so there is no risk of residual compounds.

[0219] Animal cell culture medium has a high protein content, and conventional aeration methods tend to make the high protein solution foam, which floats on top of the medium, affecting the gas exchange as well as affecting the function of the proteins. In contrast, the aeration process of the present disclosure takes place in the bio-oxygen carrying solution 2403a, and the culture medium does not foam.

[0220] In some examples, the gas exchange vessel 2400 is used with a cell culture vessel 2100 that comprises a scaffold assembly 200 as described with respect to [Fig. 2] to [Fig. 8] for use with adherent cells. This arrangement is beneficial as the scaffold assembly 200 and adherent cells would not be exposed to harsh cultivation conditions that conventional aeration methods create that may damage the scaffold assembly 200 and / or cause dislodgment of the cells adhering to the scaffold assembly, because the gas exchange vessel 2400 does not cause agitation (e.g. aeration by bubbles) of the culture medium. Any agitation is decoupled from the fluid flow of culture medium within the bioreactor system 2000 that is used for cell culture, and occurs separately in the target gas species liquid carrier 2403a. Additionally, the gas exchange vessel 2400 is provided separate from the cell culture vessel 2100, hence any impact caused by any agitation of the culture medium by the gas exchange vessel 2400 would be minimized by the time the culture medium reaches the cell culture vessel 2100.

[0221] Metabolite Management Device

[0222] The metabolite management device 2800 (also referred to as metabolite separation device 2800 in the present disclosure) is driven by potential difference. As shown in [Fig. 12], the main structure of the metabolite separation device 2800 comprises an electrophoresis tank shell 2801 made of plastic or other suitable material with insulating properties, which is divided into three parts by two semi-permeable membranes 2802, 2803 (e.g. only molecules with molecular weights of 500 Daltons or less are allowed to pass through) mounted on plastic (or other suitable material) frames fixed in place to form three chambers, first chamber 2804, second chamber 2806 and third chamber 2805.

[0223] As provided in [Fig. 12], the first chamber 2804 comprises a negative (cathode) zone and the second chamber 2806 may comprises a positive (anode) zone. In some examples, the first chamber 2804 may comprise a positive (anode) zone and the second chamber 2806 may comprise a negative (cathode) zone. A negative zone comprises one or more negative (cathode) electrodes 2808 and a positive zone comprises one or more positive (anode) electrodes 2807. The one or more electrodes may comprise a conductive material, for example the one or moreelectrodes may comprise metal electrodes and / or graphite electrodes. At the bottom of each of the first and second chambers 2804, 2806, there is a liquid outlet 2812, 2813, which is connected to a liquid outlet pipe (not shown), and the pipe is connected to an electric control switch, so that when the switch is opened, the liquid (i.e. buffer fluid) in the first and second chambers 2804, 2806 may be discharged. In some examples, a non-electric control switch or device may be used. In an example, the buffer liquid may flow out of outlets 2812, 2813 naturally by the effect of gravity. In some examples, liquid in first and second chambers 2804, 2806 may be actively pumped out by one or more liquid pumps. Each of the first and second chambers 2804, 2806 is provided with an inlet 2809, 2810, in which the first and second chambers 2804, 2806 are connected to one or more buffer fluid sources to supply the first and second chambers with one or more buffer fluids. A first buffer fluid may be provided to the first chamber 2804 and a second buffer fluid may be provided to the second chamber 2806. The first and second buffer fluids may be the same buffer fluid. The one or more buffer fluids may comprise potassium dihydrogen phosphate / disodium hydrogen phosphate. The one or more buffer fluid sources may be the same source. Flow of the one or more buffer fluids from the one or more buffer fluid sources to the first and / or second chambers 2804, 2806 may be controlled by an electric control switch, and a certain amount of buffer fluid may be automatically injected (for example by a buffer pump) into the first and second chambers 2804, 2806 every time the metabolite separation device 2800 operates. The third chamber 2805 is provided with a culture media inlet 2811 which is configured to receive culture medium with a first metabolite level and a culture media outlet 2814 which is configured to discharge culture medium with a second metabolite level. The second metabolite level is less than the first metabolite level.

[0224] When the metabolite separation device 2800 is in operation, compression type reflux pump 2300a (i.e. a culture fluid pump) operates and provides a certain amount of culture medium into the third chamber 2805 of the electrophoresis tank 2801, so that its liquid level is equal to the liquid level of the negative pole area 2804 and the positive pole area 2806. Then, the electrophoresis tank 2801 will be energized, where the positive and negative electrodes 2807, 2808 are configured to apply an electric field to at least the inflow culture fluid in the third chamber 2805 to cause positively charged components of the inflow culture fluid to move towards the negative electrode and negatively charged components of the inflow culture fluid to move towards the positive electrode. Under the action of low voltage DC, positively charged molecules (i.e. positively charged components) will move to the negative pole 2804 and negatively charged molecules (i.e. negatively charged components) will move to the positive pole 2806, but only small molecules can pass through the semi-permeable membranes 2802, 2803, and molecules with molecular weights of more than 500 Daltons will be intercepted by the semi-permeable membrane 2802, 2803. After about 5-15 minutes of energization, positively charged small molecules suchas potassium, sodium, ammonia, creatinine, uric acid, etc. are drawn to the negative zone 2804, and negatively charged small molecules such as chloride ions, phosphate ions, etc. are concentrated at the positive zone 2806 within their respective buffer fluids. Large molecules are intercepted near the semipermeable membrane 2802, 2803 in the third chamber 2805 of the electrophoresis tank 2801 and cannot pass through the membranes 2802, 2803, from the first chamber 2805 to the first and / or second chambers 2804, 2806. The size of the components that are permitted to pass through membranes 2802, 2803 may be predetermined and may depend on the use of the metabolite separate device 2800. The predetermine size of the components may depend on the properties of the semi-permeable membranes 2802, 2803, e g. size of pores.

[0225] In some examples, the electric field is continuously applied to the inflow culture fluid in the third chamber 2805. In some examples, the electric field is applied to the inflow culture fluid in the third chamber 2805 only when the culture fluid pump 2300a has pumped the inflow culture fluid to the third chamber 2805. In some examples, the electric field may be pulsed at fixed intervals and / or in certain rhythms and / or configurations of power amplitude and / or frequency to increase efficiency.

[0226] In some examples, the first, second and third chamber outlets 2812, 2813, 2814 are configured to operate between a closed and open configuration, wherein when the electric field is applied, the first, second and third chamber outlets 2812, 2813, 2814 are in the closed configuration. When the electric field is stopped or absent, the outlets 2812, 2813, 2814 may be in the open configuration. In some examples, after the electrification reaches a certain period, the electrification is stopped and the three discharge channels 2812, 2813, 2814 under the electrophoresis tank 2801 are simultaneously or sequentially opened automatically, the solutions in the positive and negative tanks 2804, 2806 enter the waste bag, and the medium in the middle section 2805 of the electrophoresis tank 2801 returns to the cell culture chamber 2100. In examples where pumps are used to control the flow of the culture fluid and / or buffer fluids, the pumps only operate to pump the respective fluids out of the first, second and / or third chambers 2804, 2805, 2806 when the electric field has stopped or is absent.

[0227] Cells produce metabolic waste during metabolism, and when too much metabolic waste accumulates, it begins to inhibit cell growth or may even lead to apoptosis (cell death). However, conventional removal of metabolic waste by dialysis relies on free diffusion, which is less efficient and requires the consumption of large amounts of dialysate. Another more common way is to use pressure as the driving force to prompt small molecule solvents and solutes to pass through the semi-permeable membrane, which is more efficient than the dialysis technology, but requires a higher pressure (0.1-0.5Mpa), which in many jurisdictions belongs to the category of dangeroushigh-pressure equipment, resulting in the restriction on the production and purchase, and strict regulations on use and operation.

[0228] Therefore, unlike known bioreactor systems which rely on free diffusion of metabolite into a dialysate separated from the culture medium by a semi-permeable membrane, or which rely on microfluidics or a special fluid circulation system to manage metabolites, the metabolite removal device 2800 is based on the principle of electrophoresis, whereby charged particles under the action of an electric field move towards an electrode opposite to their charge. Positively charged components of metabolic waste such as NH4 are concentrated at or around the negative electrode 2808, and negatively charged components such as lactic acid are concentrated at or around the positive electrode 2807, and the concentration of metabolic waste in the waste stream is much higher than that in the treated medium. In this device, large molecules (e.g., proteins) are intercepted by relying on a semi-permeable membrane 2802, 2803 such as a nanofiltration membrane or an ultrafiltration membrane, and metabolites are removed with high efficiency while maintaining useful components. The metabolite separation device 2800 is suitable to treat culture fluids in batches, ranging from about 50mL-2000mL per batch depending on the size of the cell culture vessel 2100. The metabolic waste separation system 2800 uses a low-voltage electric field to induce small molecules in the culture medium to concentrate in the separation tank, which is fast, with less waste liquid and high safety of the equipment. The metabolite removal element allows for full utilisation of large molecule components in the culture medium (e g. Epidermal Growth Factor (EGF)), reducing the amount of loss of the most expensive ingredients in the media (e g. serum and growth factors).

[0229] To maintain the ionic balance of the culture medium, the culture medium may be replenished with amino acids, glucose, inorganic salts, etc. via a replenishment pump before it is returned to the cell culture chamber 2100, in a way that reduces environmental fluctuations locally in the cell culture vessel 2100 and avoids damage to the cells or scaffold structures by bubble movement or bubble shear stress, favouring stable cell states. With reference to the bioreactor system 2000 of [Fig. 9], the culture medium is returned to the cell culture chamber 2100 via gas exchange vessel 2400 and compression liquid pump 2300’.

[0230] The tank 2801 of the metabolite separation device 2800 may be of a cylindrical, square, hourglass, or other shape, and where the inlets 2809, 2810, 2811 and outlets 2812, 2813, 2814 may be in different positions of height and position, or of different sizes to the design as shown, and where the membranes 2802, 2803 may be of a different design and position such as of a corrugated or funnel shape or at different angles to the media, or of multiple layers of differing pore size and material; and where the electrodes 2807, 2808 may be of a different position, size, composition, material and conductivity. In some examples, the membranes 2802, 2803 may beremovable. In some examples, the membranes 2082, 2803 are provided as a wall. In some examples, one or both walls 2082, 2803 comprise a ceramic, a laminate, a granule and / or suitable material with a microporous structure to allow substances of a certain molecular weight to pass therethrough to achieve the desired separation effect. In some examples, one or both walls 2802, 2803 are rigid. One or both walls 2802, 2803 may or may not comprise a membrane. One or both walls 2802, 2803 may comprise a rigid material (e.g. ceramic) and a flexible material (e g. a membrane). In some examples, one or both walls 2802, 2803 are electrically inert.

[0231] Jacket

[0232] The cell culture vessel 2100 and gas exchange vessel 2400 can be provided with a sterilization jacket 2050 (also referred to as a chamber cover in the present disclosure) that forms a sandwich between the jacket 2050 and the outer wall of the culture chamber 2100, 2400. An example of a jacket 2050 is provided in [Fig. 13]. The jacket 2050 comprises a first cover portion 2051a and a second cover portion 2051b, each of the first and second cover portions 2051a, 2051b comprising a recess and each portion removably connectable with the other to form a chamber cover cavity with their respective recesses, wherein the chamber cover cavity is configured to house the culture 2100 or gas exchange vessel 2400. The jacket 2050 can be made of soft or hard plastic, resembling a cylinder split in the middle, with a sealing structure made of soft, temperature-resistant material (e g. silicone, foamed silicone, rubber, thermoplastic polyurethane (TPU), etc.) with symmetrical grooves 2054, 2057, 2508 at an opening 2052. When in use, the cell culture or gas exchange vessel 2100, 2400 is placed into the jacket 2050 through the opening 2052, the conduits connected to the vessel 2100, 2400 are adjusted into the grooves 2054, 2057, 2058 in the opening 2052 of the jacket 2050, and the jacket 2050 is closed and secured with a locking clip 2053. When the jacket 2050 is in a closed configuration, the grooves 2054, 2057, 2058 form openings through which one or more bioreactor components or portions thereof may extend through. One or more redundant recesses 2054, 2057, 2058 of the jacket 2050 may be sealed with plugs of matching diameter.

[0233] The jacket comprises two tubes 2055, 2056 for connection to an ozone generator or for ventilation of filtered air or aerogel. Orifices of the tubes 2055, 2056 may be closed after use. The space between the sterilisation jacket 2050 and the vessel 2100, 2400 acts as a protective fluid cavity (i.e. a buffer space) so that microorganisms from the external environment cannot directly invade the vessel 2100, 2400, reducing the probability of reactor contamination. The fluid cavity may permit containment of a protective fluid, e.g. ozone or filtered air or aerogel, to minimize or prevent contamination of the chamber and / or its contents from the ambient environment.

[0234] The sterilization jacket 2050 isolates the vessel 2100, 2400 or other bioreactor components in a closed space similar to a clean room, allowing the user to perform cell cultures in a laboratory environment even if they do not have access to a standard GMP plant room with an air filtration system. This optional component is easy to use and can be used in conjunction with an ozone generator to create a mini clean-zone, or other suitable or permitted disinfectant can be sprayed internally, filtered air can then be pumped through to dry the internal spaces and achieve cleanliness.

[0235] In some examples, the jacket 2050 is configured to maintain the vessel 2100, 2400 at a desired temperature.

[0236] Liquid Incubation Chamber

[0237] The bioreactor system 1000, 2000 may comprise a media incubation device 2900 (also known as a culture medium incubation unit) as shown in [Fig. 14], The media incubation device 2900 contains immobilized lysozyme and the media incubation device 2900 is used for prewarming of the culture medium and removal of possible contaminants (mainly Gram-positive bacteria). As shown in [Fig. 14], the main feature of the media incubation device 2900 is the immobilization of lysozyme under mild conditions in a strip, sheet or spherical stationary phase, loaded in the incubator chamber 2902, which is irradiated and sterilised before the addition of lysozyme. The incubator chamber 2902 is connected to a filter 2903 via tubing to a peristaltic pump to drive the flow of liquid. Inflow culture fluid is received by the incubation chamber 2902 via an inlet where filter 2903 is provided. When in use, the culture medium is filtered once by filter 2903 into the incubator chamber 2902 for enzymatic sterilisation and preheating, and after a predetermined amount of time (e g. 10-30 minutes) of incubation, the culture medium in the incubator chamber 2902 is filtered a second time by a second filter (not shown) and enters the bioreactor system 1000, 2000 as an outflow culture fluid via an outlet of the incubation device 2900. The culture medium from the media incubation device 2900 may enter the bioreactor system 1000, 2000 via feeder port 1109, 2109. The second filter may be provided to the outlet of the incubation device 2900. The culture medium to be used is at risk of bacterial contamination during storage due to its nutrient-rich characteristics. This incubation device 2900 breaks down the cell walls of gram-positive bacteria, thus providing enzymatic sterilization, and reduces clogging of the filter membrane as the bacteria and their fragments are broken down by enzymes.

[0238] The media incubation device 2900 manufactured with natural lysozyme can kill Grampositive bacteria, reducing the risk of contamination during media supplementation or replacement, as well as simplifying maintenance by reducing clogging of media filter membranes.

[0239] In some examples, the media incubation device 2900 comprises a heater or cooler configured to heat or cool the inflow culture fluid. In some examples, the media incubation device2900 is configured to incubate the inflow culture fluid for a predetermined amount of time for temperature control of the inflow culture fluid before discharging the inflow culture fluid as the outflow culture fluid.

[0240] In some examples, the media incubation device 2900 comprises one or more baffles2901 for locating the immobilized enzymes used to enzymatically sterilize the inflow culture fluid.

[0241] It should be further appreciated by the person skilled in the art that variations and combinations of features described above, not being alternatives or substitutes, may be combined to form yet further embodiments falling within the intended scope of the present disclosure. As would be understood by a person skilled in the art, each embodiment, may be used in combination with other embodiment or several embodiments. Examples of such variations and combinations are provided as follows:• One or more liquid pumps 1300 of the bioreactor system 1000 of [Fig. 1] may comprise one or more of a compression reflux liquid pump 2300, a compression liquid pump 2300’, a peristaltic pump and a diaphragm pump. One or more liquid pumps 2300, 2300’ of the bioreactor system 2000 of [Fig. 9] may comprise one or more a peristaltic pump and a diaphragm pump, instead of compression reflux liquid pump 2300 and / or compression liquid pump 2300’.• The metabolite separation device 2800 of the bioreactor system 2000 of [Fig. 12] may be replaced by one or more of a dialyser and ultrafiltration unit. In some examples, the metabolite separation device 2800 may be absent, e.g. in the bioreactor system 1000.• The sterilization jacket 2050 of the bioreactor system 2000 of [Fig. 13] may be replaced by one or more of an ultra-clean table, biological safety cabinet and mobile sampling trolley (mobile clean shed). In some examples, the bioreactor system 1000 comprises sterilization jacket 2050 or the aforementioned alternatives.• The culture medium incubation unit 2900 of the bioreactor system 2000 of [Fig. 9] may be omitted or replaced by one or more of a microporous filter and an ultrasonic sterilizer. In some examples, the bioreactor system 1000 comprises culture medium incubation unit 2900 or the aforementioned alternatives.Certain components of bioreactor systems 1000, 2000 may have been described in the present disclosure as being positioned in a particular orientation. However, it will beappreciated that unless orientation is specified and required (e.g. for gravitation effect), such components may be positioned in a different orientation. For example, alternative to being vertical as shown, components of the bioreactor systems 1000, 2000 of [Fig.1] and [Fig. 9] can also be arranged horizontally.• Components of bioreactor systems 1000, 2000 may any suitable shape, e g. spiral, helical, plate, fin, nephron-like, or other shapes and forms.

Claims

CLAIMS1 . A bioreactor system comprising: a cell culture vessel; and a gas exchanger unit in fluid communication with the culture chamber, the gas exchanger unit comprising: a first exchange chamber configured to receive an inflow culture liquid from the cell culture vessel, a second exchange chamber configured to hold a target gas species liquid carrier comprising an amount of target gas species substantially different from an amount of target gas species in the inflow culture liquid to cause a target gas species concentration gradient between the inflow culture liquid and the target gas species liquid carrier, a wall configured to separate the first and second exchange chambers, the wall configured to be permeable to the target gas species and substantially impermeable to liquids, wherein the target gas species concentration gradient is configured to alter the amount of target gas species in the inflow culture liquid.

2. The bioreactor system according to claim 1 , wherein the target gas species comprises one or more of oxygen (O2), carbon dioxide (CO2), hydrogen (H2) and nitrogen (N2).

3. The bioreactor system according to claim 1 or 2, wherein the second exchange chamber comprises a second exchange chamber inlet to receive a gas comprising at least the target gas species for dissolution in the target gas species liquid carrier.

4. The bioreactor system according to claim 3, wherein the gas comprises air, O2, CO2, H2 and / or N2.

5. The bioreactor system according to claim 3 or 4, wherein the second exchange chamber comprises a target gas species disperser to disperse the received target gas species and increase an interfacial area between the target gas species and the target gas species liquid carrier.

6. The bioreactor system according to any one of claims 3 to 5, wherein the first exchange chamber comprises a first exchange chamber inlet positioned at a first end portion of thegas exchanger unit, and the second exchange chamber inlet is positioned at a second end portion of the gas exchanger unit, wherein the first and second end portions are opposing end portions of the gas exchanger unit.

7. The bioreactor system according to any one of claims 3 to 6, wherein the first exchange chamber comprises a first exchange chamber outlet positioned at the second end portion, proximal to the second exchange chamber inlet.

8. The bioreactor system according to any one of claims 1 to 7, wherein the target gas species liquid carrier comprises one or more of perfluorohexane, perfluorinated naphthalene, and glutaraldehyde polymerized bovine haemoglobin aqueous solution.

9. The bioreactor system according to any one of claims 1 to 8, wherein the cell culture vessel comprises an upper outlet and a lower outlet, wherein the upper outlet is further, relative to the lower outlet, from a surface upon which the cell culture vessel is supported.

10. The bioreactor system according to claim 9, wherein the bioreactor system is configured to provide a culture liquid as the inflow culture liquid to the gas exchanger unit via one or both upper and lower outlets when a volume of the culture liquid in the cell culture vessel is above a predetermined volume.11 . The bioreactor system according to claim 10, wherein the bioreactor system is configured to provide the culture liquid as the inflow culture liquid to the gas exchanger unit via the lower outlet when the volume of the culture liquid in the cell culture vessel is below the predetermined volume.

12. The bioreactor system according to any one of claims 1 to 11 , wherein the cell culture vessel comprises a scaffold assembly, the scaffold assembly comprising one or more scaffold sheets configured to support adherence of cells on one or more of its surfaces.

13. The bioreactor system according to claim 12, wherein the one or more scaffold sheets comprises a thickness of about 10 pm to about 100 pm.

14. The bioreactor system according to claim 12 or 13, wherein a 10 mm (width) by 50 mm (length) reference portion of the one or more scaffold sheets is configured to withstand a static tensile force between about 0.01 N to about 0.5 N before failure occurs, or between about 0.04 N to about 0.15 N before failure occurs.

15. The bioreactor system according to any one of claims 12 to 14, where a 10 mm (width) by 50 mm (length) reference portion of the one or more scaffold sheets is configured towithstand a dynamic tensile force of at least about 0.1 N before failure occurs.

16. The bioreactor system according to any one of claims 12 to 15, wherein the one or more scaffold sheets is extensible between about 5% to about 25% of its unextended length in response to a force applied to the one or more scaffold sheets, or between about 7% to about 21% of its unextended length in response to a force applied to the one or more scaffold sheets.

17. The bioreactor system according to any one of claims 12 to 16, wherein the one or more scaffold sheets comprises a Young’s Modulus of between about 3.0 MPa to about 7.0 MPa, or between about 3.5 MPa to about 6.5 MPa.

18. The bioreactor system according to any one of claims 12 to 17, wherein the one or more scaffold sheets comprises a tensile strength of between about 0.1 MPa to about 0.6MPa.

19. The bioreactor system according to any one of claims 12 to 18, wherein the one or more scaffold sheets comprises one or more mycelial scaffold sheets comprising intertwined mycelial fibres.

20. The bioreactor system according to claim 19, wherein the mycelial fibres are from one or more of fungal species comprising: Volvariella volvacea (Straw Mushroom), Lentinula edodes (shiitake mushroom), Hericium erinaceus (lion's mane mushroom), Ganoderma lucidum (lingzhi mushroom / reishi mushroom), Pleurotus eryngii (king oyster mushroom), Pleurotus ostreatus (oyster mushroom), Flammulina velutipes (enoki mushroom), Agaricus bisporus (button mushroom), Grifola frondosa (maitake mushroom) and Rhizopus oligosporus.

21. The bioreactor system according to claim 19 or 20, wherein the one or more scaffold sheets comprises ergothioneine.

22. The bioreactor system according to claim 21 , wherein the ergothioneine is from a fungal mycelium used to form the mycelial scaffold sheet.

23. The bioreactor system according to claim 21 or 22, where the one or more mycelial scaffold sheets comprises ergothioneine at a concentration of about 0.1 mg / g to about 15.0 mg / g of a dry mycelial scaffold sheet or about 0.2 mg / g to about 12.0 mg / g of the dry mycelial scaffold sheet.

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