Cell culture scaffolds

Abstract

The present invention relates to scaffolds for culturing animal cells and methods of preparing the scaffolds. The present invention also relates to methods of culturing cells on the scaffolds. The present invention also relates to methods of producing cell culture products and the products produced therefrom.

Description

[0001] CELL CULTURE SCAFFOLDS

[0002] FIELD

[0003] The present invention relates to scaffolds for culturing animal cells and methods of preparing the scaffolds. The present invention also relates to methods of culturing cells on the scaffolds. The present invention also relates to methods of producing cell culture products and the products produced therefrom.

[0004] BACKGROUND

[0005] Cell culture and tissue culture technologies are entering a new context with recent pursuits in a field called “Cellular Agriculture”. The concept of cellular agriculture is to produce affordable materials for human or animal consumption that previously have been procured from animals. The process removes / reduces the need for animal meat / products by cultivating animal cell directly. These materials may include foods, nutritional supplements, animal feeds, virus production, vaccine production, fast moving consumer goods or materials such as leather or cosmetics. A limiting factor in the cellular agriculture area is affordable scaffolds for cell culture. Such scaffolds are useful for increasing cell culture density, shaping the food products, increasing cell differentiation, contributing nutritional components, and texture to final products. There is also a paucity of affordable and edible scaffolds for cell culture.

[0006] It is against this background that the present inventors have developed a new scaffold for culturing animal cells and method of production thereof. The present inventors have also developed methods for producing cell culture products and products produced thereof.

[0007] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

[0008] SUMMARY

[0009] In an aspect, the present invention provides a grain comprising: i) an outer layer with at least one fracture, ii) an inner layer, and iii) at least one animal cell between the outer and the inner layer and / or at least partially in the fracture. In an embodiment, at least one fracture spans the depth of the outer layer. In an embodiment, at least one fracture provides an opening to the inner layer.

[0010] In an aspect, the present invention provides a method for preparing a scaffold for culturing animal cells comprising forming at least one fracture spanning an outer layer of a grain.

[0011] In an aspect, the present invention provides a scaffold for culturing animal cells comprising a grain having an outer layer with at least one fracture.

[0012] In an aspect, the present invention provides a method of producing a cell culture product, the method comprising: a) adding the scaffold as described herein and one or more animal cells to a cell culture apparatus; and b) culturing the cells.

[0013] In an aspect, the present invention provides a cell culture product comprising the grain as described herein, or the scaffold as described herein.

[0014] In an aspect, the present invention provides a grain comprising i) an outer bran layer with at least one fracture, ii) an inner endosperm, and iii) at least one animal cell between the bran layer and the endosperm and / or at least partially in the fracture.

[0015] In an aspect, the present invention provides a method for preparing a scaffold for culturing animal cells comprising forming at least one fracture in the bran layer of a grain.

[0016] In an aspect, the present invention provides a scaffold for culturing animal cells comprising a grain having a bran layer with at least one fracture in the bran layer.

[0017] In an aspect, the present invention provides a grain comprising a fracture in the outer layer of the grain and at least one cell at least partially in the fracture.

[0018] In an aspect, the present invention provides a scaffold for culturing animal cells comprising a grain comprising a fracture in the outer layer of the grain and at least one cell at least partially in the fracture.

[0019] In an aspect, the present invention provides a method for preparing a scaffold for culturing animal cells comprising forming a fracture in the outer layer of a grain.

[0020] In an aspect, the present invention provides a milled polished grain comprising a fracture in the outer layer of the grain and at least one cell at least partially in the fracture.

[0021] In an aspect, the present invention provides a scaffold for culturing animal cells comprising a milled polished grain comprising a fracture in the outer layer of the grain and at least one cell at least partially in the fracture.

[0022] In an aspect, the present invention provides a method for preparing a scaffold for culturing animal cells comprising forming a fracture in the outer layer of a milled polished grain. BRIEF DESCRIPTION OF THE FIGURES

[0023] Preferred embodiments of the present disclosure will be further described and illustrated, by way of example only, with reference to the accompanying drawings:

[0024] Figure 1 : Scanning electron microscopy (SEM) revealed that the outer layers of whole grain rice comprise structural features that may promote mammalian muscle cell binding. SEM of whole grain rice either (A) directly from the commercial source, or (B) following sterilization by autoclave and saline washing for 3 days is shown at various magnifications (bracketed numbers). The fracture points that facilitate myoblast access to the vacuous space between the bran and endosperm layers of the rice grain are indicated by arrows. (B) ii) is an inset of (B) i) (box indicates area). Scale bars are shown in microns.

[0025] Figure 2: Scanning electron microscopy (SEM) revealed that differentiated myotube structures actively grow from the fracture points in the bran layer of whole grain rice. SEM of processed whole grain rice that were seeded with C2C12 myoblasts and maintained under differentiation conditions for 4 weeks. Differentiated myotube structures and cellular growths are depicted in the foreground of (B) and (D) and rice structures are shown in the background of (B) and (D). Myotube outgrowths that were projecting outwards from established cellular growth beneath the bran layer of the grain are indicated by arrows. (B) is an inset of (A), and (D) is an inset of (C) (box indicates area). Scale bars are shown in microns.

[0026] Figure 3: Schematic representation of the cellular process occurring in Figure 2. Fracture points in the bran layer (1 ) of whole grain rice, the size and frequency of which are exacerbated by extensive saline washing (2), facilitate access for seeded myoblasts to access the vacuous space between the bran and endosperm layers of whole grain rice (3). This vacuous space provides a favorable environment for the myoblasts to bind to the rice structure and also provides protection from fluid shear stress of the media. Once bound the myoblasts proliferate and differentiate to the mature myotube structures shown in Figure 2 (4).

[0027] Figure 4: Scanning electron microscopy (SEM) revealed that the interior of differentiated myotube structures contained tightly packed elongated cellular structures and cylindrical vacuous structures. SEM of processed whole grain rice that were seeded with C2C12 myoblasts and maintained under differentiation conditions for 4 weeks. The differentiated myotube structure is depicted in the foreground of the image and the rice is depicted in the background of the image. An observed break in through the vertical axis of the mature myotube in the sample permitted examination of the interior of a myotube structure by SEM. The interior of the mature myotube comprised tightly packed elongated cellular structures (p) and cylindrical vacuous structures (v). (B) and (C) are insets of (A) (box indicates areas). Scale bars are shown in microns.

[0028] Figure 5: Scanning electron microscopy (SEM) revealed undifferentiated monolayer and amorphous aggregate myoblast structures growing around the fracture points in the bran layer of whole grain rice. SEM of processed whole grain rice that were seeded with C2C12 myoblasts and maintained under differentiation conditions for 4 weeks. Excluding panel C, cellular structures and cellular growths are depicted in the foreground of the images and rice structures in the background of the images. Amorphous aggregate (B, C, D, E) muscle cell structures were observed to grow from (or around) the fracture points in whole grain rice. These cellular growths can be seen extending from the vacuous space between the bran and endosperm layers of whole grain rice (arrows). (E) Shows an inset of (D) (box indicates areas). Scale bars are shown in microns.

[0029] Figure 6: Scanning electron microscopy (SEM) confirmed that white rice, lacking a bran layer, was more uniform compared to that of whole grain rice. SEM of white rice either (A) directly from the commercial source, or (B) following sterilization by autoclave and saline washing for 3 days is shown at various magnifications (bracketed numbers). Fracture points were detected in white rice following saline washing (arrow) however these were less frequent and smaller in magnitude when compared to whole grain rice. Magnifications are shown as bracketed numbers. Scale bars are shown in microns.

[0030] Figure 7: Quantification of cellular material bound to rice showed that whole grain rice is a more efficient bioscaffold than white rice for mammalian muscle cells. Whole grain (wh. gr. - shown on right) or white (wh.- shown on left) rice that were seeded with C2C12 myoblasts (or no cell controls for background quantification) were maintained under differentiation conditions for 4 weeks and were then assessed for bound cellular material by (A) Cell Titer Blue (CTB) cell viability assay or total cellular protein (B & C). Statistical significance values shown were determined by unpaired t test.

[0031] Figure 8: Processing of whole grain rice using the method described in the Rice bio-scaffolds methods section resulted in a decrease in mass. (A) Individual lots of whole grain rice were weighed (prewash) and then processed as described in Methods (n=6, dark lines and dots). Control rice was weighed, but not processed (n=1 , dotted line and dots). Processed rice samples were subjected to 3 final washes in sterile ddH2O to remove any residual salt, and then dried in a non-humidified orbital shaker maintained at 45°C for 24 hours. Dried rice and control rice samples maintained under identical conditions were then re-weighed (post wash). (B) Mean of differences between pre and post wash mass values was subjected to paired t-test analysis and was found to be significant (**, p=0.0019).

[0032] Figure 9: Quantification of viable cellular material bound to rice by Cell Titer Blue assay showed that processed whole grain rice (processed) is a more efficient bioscaffold for mammalian myoblasts than unprocessed whole grain rice (unprocessed). Rice grains that were seeded with C2C12 myoblasts (or no cell controls for background quantification) were maintained for 7 days in growth media and then extensively washed prior to cell viability assessment by Cell Titer Blue (CTB) cell viability assay. Statistical significance values were determined by unpaired t test (A) p=0.0413 and (B) p=0.0004. (B) Shows an analysis of the same data shown in (A) with the two high signal outliers in the “processed” group removed (data points removed are marked by arrows in panel (A).

[0033] DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0034] Preferred embodiments of the present invention are described below by way of example only.

[0035] Definitions

[0036] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture and serum).

[0037] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0038] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0039] As used herein, the term “about”, unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, even more preferably + / - 1 %, of the designated value.

[0040] As used herein, the phrase “at least one of”, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing , or category. In other words, “at least one of” means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0041] It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

[0042] Throughout the present specification, various aspects and components of the invention can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1 , 2, 3, 4, 5, 5.5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0043] Grain

[0044] As used herein, a “grain” is a small dry seed often harvested for animal or human consumption. Grains comprise layers, the layers of the grain have different compositions.

[0045] It will be a appreciated by a person skilled in the art that a grain of the present invention can be any grain in which introducing at least one fracture into an outer layer creates a space where at least one animal cell can be seeded between the outer layer and an inner layer and / or at least partially in the fracture.

[0046] A person skilled in the art will appreciate that the grain as described herein can be, for example, a cereal grain or a legume grain.

[0047] In some embodiments, when the grain is a cereal grain, the outer layer is the bran layer. In some embodiments, when the grain is a cereal grain, the inner layer is the endosperm. In some embodiments, when the grain is a pulse, the outer layer is a seed coat. In some embodiments, when the grain is a pulse, the outer layer is the testa. In some embodiments, the grain is a pulse the inner layer is the cotyledon.

[0048] In some embodiments, parts of the grain that are not desirable for human or animal consumption have been removed from the grain (e.g. the husk).

[0049] In some embodiments, the “outer layer” as referred to here is not the outermost layer of a grain as it exists in nature, e.g. for some cereal grains the outer layer may be surrounded by a husk or partially surrounded by a husk.

[0050] As used herein, “fracture” refers to a breakage point or opening in the outer layer of the grain. In an embodiment, the breakage point is a disruption of the native / normal structure of the layer. In an embodiment, the fractures spans the depth of the outer layer of the grain. In an embodiment, the fracture is of sufficient size to allow a cell to be seeded between the outer layer and an inner a layer of the grain and / or at least partially in the fracture. In an embodiment, the fracture is of sufficient size to allow a cell to be seeded between the outer layer and an inner a layer of the grain. In an embodiment, the fracture is of sufficient size to allow a cell to be seeded at least partially in the fracture. In some embodiments, the fracture as described herein provides access to a space and / or creates a space within the grain that is sterically favourable for an animal cell to one or more of anchor, grow, differentiate and proliferate. In some embodiments, the space is between an outer bran layer and an inner endosperm layer. In some embodiments, the space is partially in the fracture. In some, embodiments, the space is within the fracture. In an embodiment, a cell seeded between the outer layer and an inner layer of the grain and / or at least partially in the fracture is exposed to reduced fluid sheer stress from the cell culture medium during cell culture.

[0051] In some embodiments, the grain comprises one to seven fractures, or one to six fractures, or one to five fractures, or one to four fractures, or one to three fractures. In some embodiments, the grain comprises one to seven fractures. In some embodiments, the grain comprises one to six fractures. In some embodiments, the grain comprises one to five fractures. In some embodiments, the grain comprises one to four fractures. In some embodiments, the grain comprises or one to three fractures. In some embodiments, the grain comprises at least two fractures. In some embodiments, the grain comprises at least three fractures. In some embodiments, the grain comprises at least four fractures. In some embodiments, the grain comprises at least five fractures. In some embodiments, the grain comprises at least six fractures.

[0052] In some embodiments, at least one of the fractures has a width of about 50 pm to about 750 pm, or about 50 pm to about 500 pm, or about 100 pm to about 450 pm, or about 150 pm to about 400 pm, or about 200 pm to about 350 pm, or about 250 pm to about 300 pm. In some embodiments, at least one of the fractures has a width of about 50 pm to about 750 pm. In some embodiments, at least one of the fractures has a width of about 50 pm to about 500 pm. In some embodiments, at least one of the fractures has a width of about 100 pm to about 450 pm. In some embodiments, at least one of the fractures has a width of about 150 pm to about 400 pm. In some embodiments, at least one of the fractures has a width of about 200 pm to about 350 pm. In some embodiments, at least one of the fractures has a width of about 250 pm to about 300 pm.

[0053] In some embodiments, at least one of the fractures has a length of about 50 pm to about 750 pm, or about 50 pm to about 500 pm, or about 100 pm to about 450 pm, or about 150 pm to about 400 pm, or about 200 pm to about 350 pm, or about 250 pm to about 300 pm. In some embodiments, at least one of the fractures has a length of about 50 pm to about 750 pm. In some embodiments, at least one of the fractures has a length of about 50 pm to about 500 pm. In some embodiments, at least one of the fractures has a length of about 100 pm to about 450 pm. In some embodiments, at least one of the fractures has a length of about 150 pm to about 400 pm. In some embodiments, at least one of the fractures has a length of about 200 pm to about 350 pm. In some embodiments, at least one of the fractures has a length of about 250 pm to about 300 pm.

[0054] In some embodiments, when in the cell culture environment, the space is hypoxic compared to the surrounding cell culture environment which is favourable for the growth of e.g. muscle cells. In an embodiment, hypoxic in the cell culture environment refers to about 1% to about 12% O2, to about 1 % to about 10% O2, to about 1 % to about 7% O2, or about 1 % to about 6% O2, or about 2% to about 6% O2, or about 2% to about 5% O2, or about 2% to about 4% O2. In an embodiment, hypoxic refers to about 1 % to about 6% O2. In an embodiment, hypoxic refers to about 2% to about 6% O2. In an embodiment, hypoxic refers to about 2% to about 5% O2. In an embodiment, hypoxic refers to about 2% to about 4% O2. In some embodiments, when in the cell culture environment, a cell in the space is protected from sheer forces created by the movement of the medium (e.g. with one or more of shaking, stirring or perfusion).

[0055] Cereal grain

[0056] In some embodiments, the grain is a cereal grain. In some embodiments, where the grain is a cereal grain, the outer layer is a bran layer. In some embodiments, when the grain is a cereal grain, the bran is the portion of the grain which surrounds the starchy endosperm. In cereal grains the bran is surrounded by a husk (also referred to as a hull). In some embodiments, when the grain is a cereal grain, the inner layer as referred to herein is the endosperm.

[0057] In embodiments, where the grain is a cereal grain, the outer layer comprising at least one fracture as described herein is not the husk layer. In some embodiments of the present invention, the husk is fully or partially removed from the grain. In some embodiments, about 10% to about 90% of the husk is removed. In some embodiments, about 20% to about 90% of the husk is removed. In some embodiments, about 30% to about 90% of the husk is removed. In some embodiments, about 40% to about 90% of the husk is removed. In some embodiments, about 50% to about 90% of the husk is removed. In some embodiments, about 60% to about 90% of the husk is removed. In some embodiments, about 70% to about 90% of the husk is removed. In some embodiments, about 80% to about 90% of the husk is removed. In some embodiments, the husk is 100% removed from the grain.

[0058] In an embodiment, the cereal is selected from rice, wheat, barley, millet, oats, teff, rye, corn, quinoa and buckwheat.

[0059] In an embodiment, the cereal is rice. As used herein, the term “rice” refers to a species of the genus Oryza.

[0060] In preferred embodiments, the rice comprises a bran layer (also referred to herein as brown rice). In some embodiment, the rice does not comprise a bran layer (also referred to herein as white rice).

[0061] In some embodiments, the rice grain is about 2 mm to 10 mm in length. In some embodiments, the rice grain is an extra-long rice grain (about 7.5 mm to about 10 mm in length). In some embodiments, the rice grain is a long rice grain (about 6.61 mm to about 7.5 mm in length). In some embodiments, the rice grain is a medium rice grain (about 5.51 mm to about 6.60 mm in length). In some embodiments, the rice grain is a short rice grain (about 2 mm to about 5.50 mm in length).

[0062] In some embodiments, the rice grain has a width of about 2.5 mm to about 3.10 mm. In some embodiments, the rice grain has a length -to-width ratio of about 3. In some embodiments, the rice grain has a length-to-width ratio of about 2.1 to about 3. In some embodiments, the rice grain has a length-to-width ratio of about 1 .1 to about 2. In some embodiments, the rice grain has a length-to-width ratio of about 1 .

[0063] In an embodiment, the Oryza species is selected from Oryza sativa (Asian rice), Oryza glaberrima (African rice), Oryza rufipogon (common wild rice), Oryza nivara (annual wild rice), Oryza barf / ? / / (Barth's rice), Oryza longistaminata (longstamen rice), Oryza meridionalis (Australian wild rice) and Oryza glumaepatula (South American wild rice).

[0064] In an embodiment, the Oryza sp. is Oryza sativa (Asian rice). In an embodiment, the Oryza sp. is Oryza glaberrima (African rice). In an embodiment, the Oryza sp. is Oryza rufipogon (common wild rice).

[0065] In an embodiment, the Oryza sativa is selected from indica, temperate japonica, tropical japonica, aus, aromatic and interspecies crosses thereof.

[0066] In an embodiment, the Oryza sativa indica is selected from Oryza sativa indica mutica, Oryza sativa indica elongata, Oryza sativa indica atrobrunnea, Oryza sativa indica gilanica, and Oryza sativa indica adusta.

[0067] In an embodiment, the Oryza sativa japonica is selected from Oryza sativa japonica italica, Oryza sativa japonica amaura, Oryza sativa japonica malanotriz, Oryza sativa japonica malanotrix, and Oryza sativa japonica dichroa.

[0068] In an embodiment, the Oryza glaberrima is selected from Oryza glaberrima rustica, Oryza glaberrima rigida, Oryza glaberrima evolute and Oryza glaberrima aspera.

[0069] In some embodiments, the rice is a non-genetically modified rice. In some embodiments, the rice is a genetically modified rice (e.g. golden rice or golden rice 2).

[0070] In an embodiment, the cereal is wheat. As used herein, the term "wheat" refers to a species of the genus Triticum. In an embodiment, the wheat is selected from a diploid wheat, hexapioid wheat, tetrapioid wheat, einkorn and farrow. In an embodiment, the wheat is a wild wheat. In an embodiment, the wheat is a domestic wheat.

[0071] In an embodiment, the wheat is a diploid wheat. In an embodiment, the diploid wheat is selected from Triticum boeoticum, Triticum urartu, Triticum monococcum ssp. aegilopoides.

[0072] In an embodiment, the wheat is a hexapioid wheat. In an embodiment, the hexapioid wheat is selected from Triticum aestivum, Triticum spelta, Triticum macha, Triticum compactum, Triticum sphaerococcum, Triticum vavilovii, and interspecies crosses thereof. In an embodiment, the hexapioid wheat is selected from Triticum aestivum and Triticum vavilovii. In an embodiment, the Triticum aestivum is selected from Triticum aestivum compactum (club wheat), Triticum aestivum sphaerococcum (“Indian dwarf” or “shot wheat”), Triticum aestivum spelta (“spelt wheat”), Triticum aestivum macha. A preferred species of hexapioid wheat is Triticum aestivum ssp aestivum ("breadwheat").

[0073] In an embodiment, the wheat is a tetrapioid wheat. In an embodiment, the tetrapioid wheat is selected from Triticum durum (durum wheat, freekeh or Triticum turgidum ssp. durum), Triticum dicoccoides (wild emmer), Triticum dicoccum (emmer), Triticum polonicum (Polish wheat), Triticum paleocolchicum, Triticum turanicum (Khorasan wheat), Triticum carthlicum (also termed “Persian wheat”), Triticum turgidum (Rivet, cone or English wheat), Triticum araraticum, Triticum timopheevii ssp. Armeniacum, Triticum timopheevii ssp. timopheevii and interspecies crosses thereof.

[0074] In an embodiment, the wheat is einkorn wheat.

[0075] In an embodiment, the wheat is a farrow. In an embodiment, farrow is selected from Triticum spelta (spelt), Triticum dicoccum (emmer), and Triticum monococcum (einkorn).

[0076] In an embodiment, the cereal is barley. As used herein, the term "barley" refers to a species of the genus Hordeum. In an embodiment, the Hordeum species is selected from Hordeum vulgare L. ssp. spontaneum (C.Koch.)Thell, Hordeum bulbosum L. or another species suitable for commercial production of grain.

[0077] In an embodiment, the cereal is corn. As used herein, the term “corn” or “maize” refers to a species of the genus Zea. In an embodiment, the corn is selected from a flour corn, popcorn, dent corn, flint corn, sweet corn, waxy corn, amylomaize, pod corn and striped maize. In an embodiment, the corn is a cultivated corn.

[0078] In an embodiment, the Zea species is selected from Zea mays, Zea diploperennis, Zea luxurians, Zea nicaraguensis and Zea perennis.

[0079] In an embodiment, the Zea mays is a Zea mays L.

[0080] In an embodiment, the Zea mays L. is selected from Zea mays ssp. Mexicans, Zea mays ssp. cultivar:B73, Zea mays ssp. cultivar:Mo 17, Zea mays ssp. cultivar:SK, Zea mays ssp. cultivar:HZS, Zea mays ssp. cultivar:KN5585, Zea mays everta, Zea mays amylacea, Zea mays indentata, Zea mays indurata, Zea mays saccharata, Zea mays rugosa, Zea mays ceratina, Zea mays tunicata Larrahaga ex A. St. Hil. and Zea mays japonica.

[0081] In an embodiment, the cereal is millet. In an embodiment, the millet is finger millet Eleusine coracana). In an embodiment, the millet is pearl millet {Pennisetum glaucum). In an embodiment, the millet is foxtail millet Setaria italic). In an embodiment, the millet is Japanese barnyard millet (Echinochloa esculenta). In an embodiment, the millet is Indian barnyard millet (Echinochloa frumentacea). In an embodiment, the millet is Burgu millet (Echinochloa stagnina). In an embodiment, the millet is little millet (Panicum sumatrense). In an embodiment, the millet is proso millet (Panicum miliaceum). In an embodiment, the millet is sonoran millet (Panicum sonorum). In an embodiment, the millet is kodo millet (Paspalum scrobiculatum). In an embodiment, the millet is guinea millet (Brachiaraia deflexa). In an embodiment, the millet is browntop millet (Brachiaraia ramose).

[0082] In an embodiment, the cereal is a tef. In an embodiment the tef is a Poaceae.

[0083] In an embodiment, the cereal is a sorghum. In an embodiment, the sorghum is Sorghum bicolour (sorghum).

[0084] In an embodiment, the cereal is an oat. In an embodiment, the oat is an Avena. In an embodiment, the Avena is selected from Avena sativa (common oats), Avena abyssinica (Ethiopian oats).

[0085] In an embodiment, the cereal is a rye. In an embodiment, the rye is a Secale. In an embodiment, Secale is Secale cereale (rye).

[0086] In an embodiment, the cereal is quinoa. As used herein, the term “quinoa” refers to a species of the genus Chenopodium. In an embodiment, the Chenopodium is Chenopodium quinoa (quinoa).

[0087] In an embodiment, the cereal is buckwheat. As used herein, the term “buckwheat” refers to a species of the genus Fagopyrum. In an embodiment, the Fagopyrum is selected from Fagopyrum esculentum (buckwheat) and Fagopyrum tatricum (tartary buckwheat).

[0088] Legume grain

[0089] In an embodiment, the grain is a pulse from a legume. In an embodiment, the pulse is selected from a bean, lentil, pea, chickpea and soybean. In an embodiment, the pulse is a bean. In an embodiment, the pulse is lentil. In an embodiment, the pulse is pea. In an embodiment, the pulse is chickpea. In embodiment, the pulse is a soybean.

[0090] In an embodiment, when the grain is a pulse, the outer layer is a seed coat. In an embodiment, when the grain is a pulse, the outer layer is the testa. In an embodiment, the grain is a pulse the inner layer is the cotyledon.

[0091] In an embodiment, the pulse is produced by a member of the Fabaceae family. In an embodiment, the Fabaceae is selected from Phaseolus, Pisum, Vigna, Cajanus, Lens, Cicer, Vida, Arachis, Glydne, Macrotyloma, Mucuna, Lupines, Ceratonia, Canavalia, Cyamopsis, Lablab, Psophocarpus, Clitoria, Lathyrus, Trifolium, Medicago, Melilotus, and Tamarindus.

[0092] In an embodiment, the Phaseolus is selected from Phaseolus vulgaris (kidney beans), Phaseolus lunates (lima beans), Phaseolus coccineus (runner beans), Phaseolus acutifolius (tepary bean).

[0093] In an embodiment, the Pisum is Pisum sativum (green peas).

[0094] In an embodiment, the Vigna is selected from Vigna radiata (mung beans), Vigna mungo (urad), Vigna unguiculata (cowpeas), Vigna. Aconitifolia (moth beans) Vigna angu laris (adzuki beans).

[0095] In an embodiment, the Cajanus is Cajanus cajan (pigeon pea).

[0096] In an embodiment, the Lens is Lens culinaris (lentils).

[0097] In an embodiment, the Cicer is Cicer arietinum (chickpeas).

[0098] In an embodiment, the Vicia is Vicia faba (fava beans), Vicia ervilia (bitter vetch) and Vicia sativa (common vetch).

[0099] In an embodiment, the Arachis is Arachis hypogaea (peanut).

[0100] In an embodiment, the Glycine is Glycine max (soybean).

[0101] In an embodiment, the Macrotyloma is Macrotyloma uniflorum (horsegram).

[0102] In an embodiment, the Mucuna is Mucuna pruriens (velvet bean).

[0103] In an embodiment, the Lupines is selected from Lupines albus and Lupines mutabilis.

[0104] In an embodiment, the Ceratonia is Ceratonia siliqua (carob bean).

[0105] In an embodiment, the Canavalia is selected from Canavalia gladiate (sword bean) and Canavalia ensiformis (jack beans).

[0106] In an embodiment, the Cyamopsis is Cyamopsis tetragonoloba (guar bean).

[0107] In an embodiment, the Lablab is Lablab purpureus (hyacinth bean).

[0108] In an embodiment, the Psophocarpus is Psophocarpus tetranoglobulus (winged bean).

[0109] In an embodiment, the Clitoria is Ciltoria ternatea (butterfly pea).

[0110] In an embodiment, the Lathyrus is selected from Lathyrus sativus (grass pea) and Lathyrus tuberoses (tuberous pea).

[0111] Cells The skilled person would appreciate that the cells of invention can be any cell which can be cultured in vitro on a grain or scaffold as described herein.

[0112] In some embodiments, the cell is from a continuous cell line. In some embodiments, the cell is from a primary cell line. In some embodiments, the cell is from an immortalized cell line (e.g. as described in Soice and Johnston, 2021 ).

[0113] In an embodiment, the immortalised cell line is a spontaneous immortalised cell line. In an embodiment, the immortalised cell line does not comprise an oncogene.

[0114] In some embodiments, the cell line is isolated from a tumor.

[0115] In some embodiments, the cell line is modified with a footprint free genetic modification (e.g. CRISPR). In some embodiments, the cell line is not genetically modified.

[0116] In some embodiments, the cell is an adherent cell. In some embodiments, the cell is a suspension cell. In some embodiments, when the cell is cultured in plate or flask cell culture without shaking the cell is a non-adherent cell (suspension cell).

[0117] In an embodiment, the cell is approved for safe production as a food. In an embodiment, the cell can proliferate and differentiate efficiently at industrial scales. In some embodiments, the cell can be cultured in non-animal derived reagents.

[0118] In an embodiment, the cell is an animal cell. In an embodiment, the animal is a non-human animal.

[0119] In an embodiment, the animal cell is a stem cell, a differentiated cell or a combination thereof.

[0120] In an embodiment, the animal cell is a stem cell. In an embodiment, the stem cell is an embryonic stem cell, a mysosatellite stem cell, a mesenchymal stem cell, adipose- derived stem cell, pluripotent stem cell or a combination of two or more thereof. In an embodiment, the stem cell is an embryonic stem cell. In an embodiment, the stem cell is a mysosatellite cell. In an embodiment, the stem cell is a mesenchymal stem cell. In an embodiment, the stem cell is an adipose-derived stem cell. In an embodiment, the stem cell is a pluripotent stem cell.

[0121] In an embodiment, the animal cell is a differentiated cell. In an embodiment, the differentiated cell is a myocyte, adipocyte, chondrocyte, fibroblast, epithelial, endothelial cell, extracellular matrix-producing supporting cell, or a combination of two or more thereof. In an embodiment, the animal is a myocyte. In an embodiment, the animal cell is an adipocyte. In an embodiment, the animal cell is a chondrocyte. In an embodiment, the animal cell is a fibroblast. In an embodiment, the cell is an epithelial cell. In an embodiment, the animal cell is an endothelial cell. In an embodiment, the animal cell is an extracellular matrix-producing supporting cell. In an embodiment, the animal cell is a differentiated cell derived from culturing the grain comprising a stem cell. In some embodiments, the cell differentiates into a myofiber or myotube. In some embodiments, the cell differentiates into a myotube. In some embodiments, the cell differentiates into a myofiber. In an embodiment, the animal cell is from a land-dwelling animal. In an embodiment, the animal cell is from a water dwelling animal. In some embodiments, the animal cell is from a livestock animal.

[0122] In an embodiment, the animal cell is from a warm-blooded animal. In an embodiment, the warm blood animal is a quadruped.

[0123] In some embodiments, the warm-blooded animal is a mammal or avian. In some embodiments, the warm-blooded animal is a mammal. In some embodiments, the mammal is selected from cow, sheep, buffalo, horse, bison, caribou, goat, horse, kangaroo, moose and pig. In some embodiments, the warm-blooded animal is an avian. In an embodiment, the avian is selected from a chicken, duck, emu, goose, guinea hen, ostrich, partridge, pheasant, quale, squab and turkey.

[0124] In an embodiment, the animal is a cold-blooded animal. In some embodiments, the cold-blooded animal is a fresh water animal. In some embodiments, the coldblooded animal is a salt water animal. In some embodiments, the cold-blooded animal is selected from a fish, arthropod, reptile and amphibian.

[0125] In some embodiments, the cold-blooded animal is a fish. In some embodiments, the fish is a diadromous fish. In some embodiments, the fish is an anadromous fish. In some embodiments, the fish is a catadromous fish.

[0126] In an embodiment, the fish cell is selected from a red muscle cell, pink muscle cell and a white muscle cell or a combination of two or more of the aforementioned cells.

[0127] In an embodiment, the fish is selected from salmon, tuna, carp, barbel, basa, cod, trout, smelt, sardine, barramundi, snapper, catfish, perch, perch-like fishes, milkfish and halibut. In an embodiment, the fish is a cyprindae.

[0128] Examples of fish cell lines suitable for seeding on the grain or scaffold as described herein are described for example in Rubio et al (2019).

[0129] In some embodiments, the cold-blooded animal is a shellfish. In an embodiment, the shellfish is a Crustacea. In an embodiment, the Crustacea is selected from a shrimp, prawn, crayfish, crab, crawfish and lobster.

[0130] In some embodiments, the shellfish is a mollusk. In an embodiment the mollusk is selected from an oyster, abalone, scallop, octopus, squid, clam, cockle, mussel and arkshell. Examples of fish and shellfish cell lines suitable for seeding on the grain or scaffold as described herein are described for example in Rubio et al. (2019).

[0131] In some embodiments, the cold-blooded animal is an arthropod. In some embodiments, the cold-blooded animal cell is reptile. In some embodiments, the coldblooded animal is an amphibian.

[0132] In some embodiments, cells from two or more animals are cultured on the grain or scaffold as described herein.

[0133] Method for preparing a scaffold

[0134] In an aspect, the present invention provides a method for preparing a scaffold for culturing animal cells comprising producing at least one fracture spanning an outer layer of a grain wherein the fracture provides an opening to an inner layer of the grain . In an embodiment, when the grain is a cereal grain, and the outer layer is the bran layer and the inner layer is an endosperm. In an embodiment, when the grain is a pulse from a legume the outer layer is the seed coat or the testa and the inner layer is the cotyledon.

[0135] In an embodiment, the method reduces the net mass of the grain. In an embodiment, the net mass of the grain is reduced by about 0.05% to about 2%. In an embodiment, the net mass of the grain is reduced by about 0.08% to about 1 .8%. In an embodiment, the net mass of the grain is reduced by about 1 % to about 1.5%. In an embodiment, the net mass of the grain is reduced by about 1 % to about 1 .2%.

[0136] In an embodiment, the method comprises removing a non-soluble component from the grain. In an embodiment, the method comprises removing starch from the grain. In an embodiment, the starch is removed from between the inner and the outer layer as described herein. In an embodiment, the method comprises removing arsenic from rice. In an embodiment, the method comprises removing the husk or a part thereof from the grain.

[0137] In an embodiment, the method comprises a) detoxifying the grain; b) sterilizing the grain; and c) washing the sterilised grain, wherein steps a), b) and c) combined produce at least one fracture spanning the bran layer of a grain. As used herein, detoxifying the grain refers to removing a component from the grain that negatively influences culturing a cell on a grain (e.g. removing a component from the grain that is toxic to cell culture). In an embodiment, detoxifying the grain comprises removing arsenic from the grain. In an embodiment, detoxifying the grain comprises removing excess starch from the grain. In an embodiment, sterilizing the grain comprises one or more of autoclaving, alcohol-based sterilization (e.g. 80% alcohol) and UV-light sterilization. In an embodiment, washing the sterilized grain comprises washing in phosphate buffered saline (PBS). In an embodiment, washing the sterilized grain comprises washing with a buffered aqueous solution comprising NaCI. In an embodiment, washing the sterilized grain comprises washing with a buffered aqueous solution comprising NaCI at a concentration of about 0.9% w / v. In an embodiment, it is the combination of steps a), b) and c) that produces a fracture as described herein. In an embodiment, it is the combination of steps a), b) and c) that produces a space in a grain as described herein.

[0138] In an embodiment, the method comprises treating the grain with one or more of: a) low pH; b) high pH; c) sonication; d) high salt; e) low temperature; f) high temperature; and g) physical disruption to induce at least one fracture in the outer layer of the grain. In an embodiment, the method comprises two or more of a) to g).

[0139] In an embodiment, the method comprises treating the grain with low pH. In an embodiment, the low pH is a pH of about 2 to about 5.5. In an embodiment, the low pH is a pH of about 2 to about 4. In an embodiment, the low pH is a pH of about 2 to about 3.5. In an embodiment, the low pH is a pH of about 2 to about 3.

[0140] In an embodiment, the method comprises treating the grain with high pH. In an embodiment, the high pH is a pH of about 8 to about 1 1 . In an embodiment, the high pH is a pH of about 8.5 to about 10.5. In an embodiment, the high pH is a pH of about 9 to about 10.

[0141] In an embodiment, the pH adjusted with one or more buffers for example tris(hydroxymethyl)aminomethane, phosphate, glycine, carbonate, and a non-buffered acid (e.g. 0.1 -1 M HCI) or base (0.01 -1 M NaOH).

[0142] In an embodiment, the method comprises treating the grain with sonication. In an embodiment, sonication is probe based sonication. In an embodiment, the grain is treated with sonication for about 2 to about 6 minutes, or about 3 to about 5 minutes, or about 3.5 to about 4.5 minutes, or about 3 minutes, or about 4 minutes or about 5 minutes.

[0143] In an embodiment, the method comprises treating the grain with high salt. In an embodiment, the NaCI concentration is greater than 0.2M. In an embodiment, the NaCI concentration is from 0.2M to about 5M. In an embodiment, the NaCI concentration is from 0.5M to about 5M. In an embodiment, the salt is NaCI at a concentration of about 1 M to about 5M. In an embodiment, the salt is NaCI at a concentration of about 2M to about 5M. In an embodiment, the salt is NaCI at a concentration of about 3M to about 5M. In an embodiment, the salt is NaCI at a concentration of about 4M to about 5M. In an embodiment, the method comprises treating the grain with low temperature (freeze thawing). In an embodiment, the grain is treated with low temperature 1 to 6 times, or 1 to 5 times, or 1 to 4 times, or 1 to 3 times, or 1 to 2 times. In an embodiment, low temperature is about -20°C to about -196°C. In an embodiment, low temperature is about -20°C to about -80°C. In an embodiment, low temperature treatment comprises snap freezing with liquid nitrogen (-196°C). In an embodiment, low temperature is about -80°C. In an embodiment, low temperature is about -20°C.

[0144] In an embodiment, the method comprises treating the grain with high temperature. In some embodiments, the rice is treated with a high temperature for a period of time that does result in cooking of the rice grain. For example, high temperature treatment comprises treatment at about 50°C for about 10 minutes.

[0145] In embodiment, the treatment is a high temperature short time treatment (HTST) also referred to as “flash pasteurization” or “high temperature short time pasteurization”. HTST reduces the presence of microorganisms which cause the product to degrade. In an embodiment, HTST is at about 80°C to about 121 °C. In an embodiment, HTST is at about 90°C to about 110°C. In an embodiment, HTST is at about 95°C to about 105°C. In an embodiment, HTST is at about 100°C. In an embodiment, HTST is for about 2 to about 180 seconds. In an embodiment, HTST is at about 100°C. In an embodiment, HTST is for about 2 to about 120 seconds. In an embodiment, HTST is at about 100°C. In an embodiment, HTST is for about 5 to about 60 seconds. In an embodiment, HTST is for about 5 to about 50 seconds. In an embodiment, HTST is for about 5 to about 40 seconds. In an embodiment, HTST is for about 5 to about 30 seconds. In an embodiment, HTST is for about 10 to about 20 seconds. In an embodiment, HTST is for about 12 to about 18 seconds. In an embodiment, HTST is for about 15 seconds.

[0146] In an embodiment, the method comprises treating the grain with mechanical disruption. In an embodiment, the mechanical disruption is vortexing or centrifugation (beads may be added during vortexing or centrifugation). In an embodiment, mechanical disruption comprises mechanical homogenization.

[0147] Scaffold for culturing animal cells

[0148] In an aspect, the present invention provides a scaffold for supporting animal cell growth. The scaffold provides an environment for animal cells to anchor, grow, differentiate and / or proliferate. In some embodiments, the scaffold protects the cells from the forces in the cell culture medium during cell culture that can damage the integrity of the cell.

[0149] In some embodiments, the scaffold is edible. In some embodiments, the scaffold contributes carbohydrates and micronutrients to the final cell culture product as described herein. In an embodiment, the scaffold contributes flavour to the final cell culture product as described herein. In an embodiment, the scaffold contributes texture to the final cell culture product as described herein. In some embodiments, the scaffold assists with forming the shape of the final cell culture product as described herein.

[0150] In an aspect, the scaffold comprises a grain as described herein. In an embodiment, the grains are arranged on a solid support structure. In some embodiments, the grains may be in connection with one or more other grains. As used herein, “connection” means linked or associated with. In some embodiments, the grains may be associated with one or more other grains by one or more of protein, carbohydrateor high viscosity cell culture medium. In some embodiments, the grains may be connected with one another by one or more of protein, carbohydrate and high viscosity cell culture medium.

[0151] Cell culture product

[0152] In an aspect, the present invention provides a cell culture product comprising the grain as described herein, or the scaffold as described herein. In an embodiment, the product is edible. In an embodiment, the product is a food or nutritional supplement. In an embodiment, the product is a meat substitute. In an embodiment, the product is a substitute for chicken, fish, beef, or a lamb meat product. In an embodiment, the product is a no animal harm product. In an embodiment, the product is a cultured meat product. In an embodiment, the product mimics a property of a traditional slaughtered meat. In an embodiment, the property is selected from the group consisting of taste, texture, size, shape, topography, and combinations thereof. In an embodiment, the food or nutritional supplement is for human consumption. In an embodiment, the food or nutritional supplement is for animal consumption e.g. livestock or a domesticated animal.

[0153] In an embodiment, the product is not genetically modified.

[0154] In an embodiment, the product is a fast-moving consumer product. In an embodiment, the product is a leather. In an embodiment, the product is a cosmetic. In an embodiment, the product has one or more of the following traits: desirable taste, desirable texture and desirable nutritional characteristics. In an embodiment, the product is pathogen free. In an embodiment, the product is sterilised e.g. by HTST.

[0155] A person skilled in that art would appreciate that the product may be cooked or dehydrated using food processing skills known in the art. The cooked or dehydrated product does not comprise any live animal cells.

[0156] In aspect the present invention provides a cell culture product comprising the cells grown on the grain as described herein or the scaffold as described herein (the cells have been separated or partially separated from the grain or scaffold). In some embodiments, the cells are suitable for virus replication for vaccine production. In some embodiments, the cells are suitable for a medical application.

[0157] Methods of producing a cell culture product

[0158] In an aspect, the present invention provides a method of producing a cell culture product, the method comprising: a) adding the scaffold of any one of claim as described herein and one or more animal cells to a cell culture apparatus; and b) culturing the cells.

[0159] In an embodiment, the method comprises a cell culture expansion and a cell culture production phase. The expansion and the production phase may occur in the same or in different cell culture apparatus. In an embodiment, the cell culture method is selected from batch fed-batch, continuous or perfusion cell culture. In an embodiment, the cell culture apparatus is a bioreactor. Such bioreactors will be known to the skilled person and are described for example in Alan et al. (2019). In an embodiment, the cell culture apparatus is a stirred bioreactor. In an embodiment, the cell culture apparatus is a rocking / wave bioreactor. In an embodiment, the cell culture apparatus is a spinner flask. In an embodiment, the bioreactor is a packed bed bioreactor. In an embodiment, the bioreactor is a fluidized bed bioreactor. In an embodiment, the bioreactor is a membrane bioreactor. In an embodiment, the cell culture apparatus is a perfusion bioreactor. In an embodiment, the bioreactor is a single use bioreactor. In an embodiment, the cell culture apparatus is a cell culture flask or plate.

[0160] In an embodiment, the bioreactor has a low sheer stress (a low sheer stress is described for example in Zhan et aL, 2020).

[0161] In an embodiment, the volume of the cell culture apparatus is from about 0.5L to about 6,000L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 4,000L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 3,000L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 2,000L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 1 ,000L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 500L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 250L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 150L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 100L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 50L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 25L. In an embodiment, the volume of the cell culture apparatus is from about 1 L to about 10 L.

[0162] The cells as described herein can be cultured in any cell culture medium that allows the expansion of the cells as described herein in vitro. Such mediums and processes will be known to the skilled person (see, for example, Allan et aL, 2019; Rubio et al. , 2019; Specht, 2020). Exemplary cell culture mediums for culturing the population of cells of the present invention include, but are not limited to: Iscove’s medium, UltraCHO, CD Hybridoma serum free medium, episerf medium, MediV SF103 (serum free medium), Dulbecco’s modified eagle medium (DMEM), Eagles Modified Eagle Medium (EMEM), Glasgow’s modified eagle medium (GMEM), SMIP-8, modified eagle medium (MEM), VP-SFM, ESM1 , DMEM based SFM, DMEM / F12 and DMEM / Ham’s F12 (Genzel et aL, 2009). It will be appreciated by persons skilled in the art that such mediums may be supplemented with additional growth factors, for example, but not limited, amino acids, hormones, vitamins, minerals and serum. In preferred embodiments, the growth factors and / or homones are recombinant. Optionally, such mediums may be supplemented with serum, for example foetal calf serum. In preferred embodiments, the medium is not supplemented with serum. In preferred embodiment, the medium is not supplemented with foetal calf serum.

[0163] In some embodiments, the medium is supplemented with a non-animal derived serum alternative.

[0164] In some embodiments, the cell culture medium is an animal free cell culture medium. Animal-free media and their components are never exposed to potential contamination by animal products at any point during their manufacturing . Exemplary animal free cell culture mediums include Essental 8 (Gibco), TeSR-E8 (Stem Cell Technologies cat no. 05990) and ExCellerate™ iPSC Expansion Medium (Bio-Techne).

[0165] In an embodiment, the cell culture medium comprises DMEM / F12 basal medium (the composition of which is described in Specht, 2020), ascorbic acid 2-phosphate, NaHCOs, sodium selenite, insulin, transferrin, FGF-2 and TGF- b§. The aforementioned proteins may be recombinant produced. Other proteins e.g. interleukins and cytokines may be added to the cell culture medium to induce cell differentiations. Such proteins may also be recombinantly produced.

[0166] In further preferred embodiments, the medium does not comprise and is not supplemented with an antibiotic. In an embodiment, the medium is xeno free.

[0167] In some embodiments, the medium is a high viscosity cell culture medium. The viscosity of the medium may be increased by the addition of e.g. glycerol and / or Ficoll400. In some embodiments, the viscosity of the cell culture medium is increased by the addition of one or more of carbohydrates, and glycerol.

[0168] In some embodiments, the high viscosity cell culture medium has a viscosity greater than 1 g / cm3. In an embodiment, the high viscosity cell culture medium has a viscosity of 1 .01 g / cm3to 1 .4 g / cm3. In an embodiment, the high viscosity cell culture medium has a viscosity of 1 .01 g / cm3to 1 .3 g / cm3. In an embodiment, the high viscosity cell culture medium has a viscosity of 1 .02 g / cm3to 1 .25 g / cm3. In an embodiment, the high viscosity cell culture medium has a viscosity of 1 .02 g / cm3to 1 .2 g / cm3.

[0169] EXAMPLES

[0170] The present invention is further described below by reference to the following non-limiting Examples.

[0171] Example 1 : Methods

[0172] Unless otherwise stated all reagents were obtained from Merck KGaA (Darmstadt, Germany).

[0173] Cell Culture - The C2C12 mouse myoblast cell line (Yaffe and Saxel 1997) was obtained from the European Collection of Authenticated Cell Cultures (ECACC) distributed through Merck. C2C12 cells were maintained in DMEM containing 10% v / v fetal calf serum (growth media) at 37°C / 5% CO2 in a humidified incubator and routinely tested to confirm the absence of mycoplasma contamination (Ramaciotti Centre for Genomics, UNSW, Australia). To induce myoblast fusion and differentiation of the line to the myotube phenotype growth media was replaced with DMEM containing 2% horse serum (differentiation media). When C2C12 cells were seeded on to rice scaffolds, antibiotics were added to the media (100 U / mL penicillin, 0.1 mg / mL streptomycin). Rice bio-scaffolds - White rice (jasmine long grain) or whole-grain brown rice (brown basmati) were obtained from Coles Supermarkets (Sydney, Australia). Dry rice lots were autoclaved to sterilize and then washed repeatedly for 3 days in sterile isotonic saline on an orbital rotating platform. Washed, sterile rice was packed in to a 50 mL Corning® mini bioreactor tube (a packed volume of 2-3 cm from bottom of tube was used). C2C12 myoblasts (1 x106) were seeded on to the rice bed in growth media (2-3 bed volumes of media). This process was repeated four more times at 24-hour intervals (five total seeding events). Seeded bioreactor tubes were maintained in growth media up to 7 days and then changed to differentiation media for 3 weeks. The experimental time course (4 weeks total incubation) has been demonstrated appropriate for C2C12 myotubes under static culture conditions (Rybchyn et aL, 2021 ). Media was changed every 2-3 days, at which point slight acidification of the media was often observed. Control bioreactor tubes were maintained simultaneously with rice under identical conditions without seeded cells for all experiments.

[0174] Cell Titer Blue® cell viability assays- Following 2 weeks incubation of the seeded bioreactor tubes under differentiation conditions the rice scaffolds were washed 5 times with PBS pH 7.2 with complete aspiration of each wash cycle. Post washing the entire rice beds were emptied out of the 50 mL bioreactor tubes and 24 individual rice grains were chosen at random and placed into individual wells of a 96 well plate and 200 mL growth media containing 10% (v / v) Cell Titer Blue® dye (Promega, Wl, USA). The plate was incubated at 37°C / 5% CO2 for 2 h and subsequently the contents of each well were transferred to a black-well plate and the fluorescence (560ex / 590em) was quantified using a CLARIOstar Plus microplate reader (BMG Labtech, Ortenberg, Germany).

[0175] Total cellular protein quantification assays - Following 2 weeks incubation of the seeded bioreactor tubes under differentiation conditions (or no cell controls held under identical conditions for assay background determination) the rice scaffolds were washed 5 times with PBS pH 7.2 with complete aspiration of each wash cycle. Post washing the entire rice beds were emptied out of the 50 mL bioreactor tubes and washed rice was either assayed as individual grains in individual wells of a 96 well plate or combined at random and placed 1.5 mL Eppendorf tubes (n=3). Quantification of individual rice grains by bicinchoninic acid assay (BCA) often resulted in detergent-soluble protein levels below the limit of sensitivity of the assay, and therefore the random pooled grain method was also employed for the study. For the individual grain assays a single rice grain was chosen at random and placed in an individual well of a 96 well plate. PBS pH 7.2 containing 1 % v / v Triton X- 100 (200 mL) was added to the well to release detergent soluble protein on an orbital shaking platform for 1 hour at RT and the supernatant was subsequently centrifuged (10,000 x g, 5 min). The protein concentration of the supernatant was then determined by BCA assay alongside a bovine serum albumin (BSA) standard of known concentration.

[0176] For the pooled assays, rice in each tube was weighed (ranged from 300-500 mg per tube) and the detergent soluble protein was then released by the addition of PBS pH 7.2 containing 1 % v / v Triton X-100. Tubes were vigorously vortexed prior to centrifugation (10,000 x g, 5 min). The protein concentration of the supernatant was then determined by BCA assay alongside a BSA standard of known concentration.

[0177] Absorbances of the reacted samples were measured at 562 nm using a CLARIOstar Plus microplate reader.

[0178] Scanning Electron Microscopy - Scanning electron microscopy (SEM) of rice scaffolds was carried out as previously described (Rybchyn et al 2021 ). Briefly, rice samples were fixed at 4 °C in 2.5% w / v glutaraldehyde (pH 7.4), dehydrated using a graded series of ethanol followed by Hexamethyldisilizane (HMDS). Fixed rice grains were mounted onto SEM stubs, platinum coated and viewed using an FEI Nova NanoSEM 230 (Oregon, USA) operating at 5kV.

[0179] Grayscale to colour image production - Greyscale SEM images were converted to an RGB colour format to aid visualization. Greyscale SEM images in .tiff format were acquired from the FEI Nova NanoSEM 230. Images were imported into Adobe Photoshop version 23.3.2 (Adobe, CA, USA) and converted to RGB format. The neural filter “Colorize” was used to process the greyscale image to a coloured reproduction with the following manually assigned colours in hexadecimal format (“bb2121” for cellular deposits, and “c9a61 b” for rice). Colour shading and intensity of colour was all performed automatically by the “Colorize” plugin. Cellular material was clearly identifiable compared to rice starch based on the presence of microvilli in either monolayer or myotube cellular structures. Amorphous cellular deposits, and cell filipodia were also found associated with these two major structures. The exterior side of the bran layer of rice comprised a smooth multilayered structure under SEM, and the interior side of the bran layer was “rough” and less defined. The rice endosperm comprised a honeycomblike structure on its exterior surface with no clear layered structure. Statistical analyses - GraphPad Prism 9.2 was used for all statistical analyses (unpaired t test).

[0180] Example 2: Examination of the surface structure of whole grain rice by scanning electron microscopy reveals fracture points in the outer layer that were increased by extensive washing

[0181] SEM analysis showed that the surface layer of whole grain rice contained fracture points in the outer bran layer providing sterically favorable binding sites for mononuclear mammalian myoblasts (Figure 1 A). Moreover, the extensive saline washing that was performed on whole grain rice to leech out excess starch prior to cell seeding increased the incidence and size of these fracture points (Figure 1 B). The fracture points appeared to facilitate access to the space between the endosperm and outer bran layers of the rice (Figure 1 B arrows). The fracture points varied in size up to -approximately 500 microns width and several mm in length and were therefore predicted to easily facilitate cell entry to the space between the bran and endosperm layers.

[0182] Example 3: Examination of the mass change associated with processing whole grain rice reveals a net mass change in processed whole grain rice

[0183] Individual lots of whole grain rice were weighed (prewash) and then processed as described above in the Rice bio-scaffolds methods section (n=6, dark lines and dots) (Figure 8A). Control rice was weighed, but not processed (n=1 , dotted line and dots). Processed rice samples were subjected to 3 final washes in sterile ddH2O to remove any residual salt, and then dried in a non-humidified orbital shaker maintained at 45°C for 24 hours. Dried rice and control rice samples maintained under identical conditions were then re-weighed (post wash). Mass change due to processing using the method described above equated to approximately 2% and was significant (p=0.0019 by paired t test analysis) (Figure 8B). Rice samples were weighed pre and post wash to establish that processing was associated with a net mass loss. As expected, no mass change was detected for control rice. It is anticipated that a component of the net mass loss is starch.

[0184] Example 4: Differentiated myotubes grew out of the fracture points in whole grain rice as revealed by scanning electron microscope Following several rounds of C2C12 myoblast seeding on to washed whole grain rice and maintenance under differentiation conditions for 4 weeks, SEM was carried out to reveal any cellular attachment and growth. SEM analysis showed that large C2 myotube structures (estimated size 1 -2 mm length) were actively growing out of the fracture points in whole grain rice (Figure 2 top and bottom). Where SEM was able to scan on an angle to elucidate within the fracture points of the rice grain it was clear that myotube outgrowths were projecting out from established cellular growth beneath the bran layer of the grain (Figure 2A, B, C white arrows). This observation aligned with the inventors initial hypothesis of a favorable cell seeding environment between the bran and endosperm layers of whole grain rice, as depicted schematically in Figure 3.

[0185] In some instances, myotubes were found to be cut along the vertical axis of the elongated structure (Figure 4). This break most likely occurred during sample processing, but may also of occurred due to movement of the rice grains during media exchange over the course of the experiment. This circumstance permitted SEM analysis to view the interior of the cultured myotube, showing the interior comprising both tightly packed elongated cellular structures (Figure 4, “p”) and also cylindrical vacuous structures (Figure 4, “v”) within the myotube. Microvilli present throughout the interior and exterior of the structure and can be clearly observed at high magnification (Figure 4 B and C).

[0186] Example 5: Undifferentiated monolayer and amorphous aggregates of muscle cells were attached to whole grain rice as revealed by SEM

[0187] In addition to differentiated myotube structure, monolayer cell deposits and amorphous cellular aggregates were also present on the myoblast-seeded whole grain rice following 4 weeks incubation under differentiation conditions. As shown by SEM these cellular deposits were typically observed at or around the fracture points within the grain or where the bran layer had been partially disrupted (Figure 5). In cases where it could be observed, the original seeding point for these structures often appeared to reside below, or were held in place by, fracture points in the bran layer (Figure 5, white arrows) as was the case for the differentiated myotubes described above (Figure 2).

[0188] Example 6: White rice was a less effective bioscaffold for muscle cells than whole grain rice SEM analysis confirmed the absence of a bran layer was lacking in our white rice sample (Figure 6). Prior to washing, the physical structure of white rice (Figure 6A) was more uniform compared to that of whole grain rice (Figure 1 A) due to the absence of the bran layer and variations in this structure. Following washing some small fracture points were present in white rice (Figure 6B arrow) however these were less frequent and smaller in magnitude when compared to whole grain rice (Figure 1 B, arrows). In summary, SEM showed that post-washing the surface structure of white rice was less variable than whole grain rice with smaller fracture points.

[0189] Quantification of cellular material that was retained on white and whole grain rice was measured using resazurin dye cell viability assay (Cell Titer Blue™; CTB) and total cellular protein (BCA assay). Four weeks post C2C12 myoblast seeding and maintenance under differentiation conditions, individual grains of rice (n=24) were assessed and whole grain rice showed significantly higher levels of cell viability by CTB assay compared to white rice (Figure 7A, p=0.0014) and total cellular protein by BCA assay (Figure 7B, p=0.0003). Where rice grains were pooled prior to BCA assay, to increase the soluble protein concentration of the sample, whole grain rice again showed increased levels of bound total cellular protein (Figure 7C, p=0.0098).

[0190] Example 7: Processed whole grain rice is more effective as a bio-scaffold for myoblast attachment than unprocessed whole grain rice

[0191] Quantification of viable cellular material bound to rice by Cell Titer Blue assay showed that processed whole grain rice (processed) is a more efficient structure for mammalian myoblast attachment than unprocessed whole grain rice (unprocessed) (Figure 9). Rice grains that were seeded with C2C12 myoblasts (or no cell controls for background quantification) were maintained in growth media as described above in the methods section (except were cultured for 7 days instead of 4 weeks to allow assessment of attachment) and then extensively washed prior to cell viability assessment by Cell Titer Blue (CTB) cell viability assay. Greater attachment of cells was observed to whole grain rice processed compared to unprocessed whole grain rice. 92% of the grains on the processed rice, and 12.5% of the grains on unprocessed rice were above the absolute threshold of sensitivity of the assay, and 75% of the grains on the processed rice, and 0% of the grains on unprocessed rice were greater than 1 standard deviation above the threshold of sensitivity. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. This application claims priority from Australian Provisional Application No.

[0192] 2023902318 entitled “Cell culture scaffolds” filed on 20 July 2023, the entire contents of which are hereby incorporated by reference.

[0193] All publications discussed and / or referenced herein are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0194] LIST OF REFERENECES

[0195] Allan et al (2019) Front. Sustain. Food Syst., Vol 3 Article 4 doi.org / 10.3389 / fsufs.2019.00044 Rubio et al (2019) Front. Sustain. Food Syst. Vol 3 Article 43 doi.org / 10.3389 / fsufs.2019.00043 Rybchyn et al (2021) ACS Omega. 6(49) 33923-31 doi.org / 10.1021 / acsomega.1c05235 Solee and Johnston (2021) Int. J. Mol. Sci. 22(21) 11660 doi.org / 10.3390 / ijms222111660 Specht (2020) An analysis of culture medium costs and production volumes for cultivated meat. The Good Food Institute.

[0196] Yaffe and Saxel (1977) Nature, (270) 725-27 doi.org / 10.1038 / 270725a0

[0197] Zhan et al (2020) IScience Nov 20; 23(11) 101653. doi: 10.1016 / j .isci.2020.101653

Claims

CLAIMS:1 . A grain comprising i) an outer layer with at least one fracture, ii) an inner layer, and iii) at least one animal cell between the outer and the inner layer and / or at least partially in the fracture.

2. The grain of claim 1 , wherein the animal cell is a stem cell.

3. The grain of claim 1 or claim 2, wherein the stem cell is an embryonic stem cell, a mysosatellite cell, a mesenchymal stem cell or a combination of two or more thereof.

4. The grain of any one of claims 1 to 3, wherein the animal cell is a myocyte, adipocyte, chondrocyte, or a combination of two or more thereof.

5. The grain of any one of claims 1 to 4, which comprises a combination of stem cells and differentiated cells.

6. The grain of any one of claims 1 to 5, wherein the cell is a differentiated cell derived from culturing the grain comprising a stem cell.

7. The grain of claim 5 or claim 6, wherein the grain is in connection with one or more further grains by the differentiated cell.

8. The grain of any one of claims 1 to 7, wherein the cell forms a myotube.

9. The grain of any one of claims 1 to 8, wherein the animal cell is from a warm blooded or a cold-blooded animal.

10. The grain of any one of claims 1 to 9, wherein the warm-blooded animal is selected from one or more of: a) a mammal; b) an avian; andc) a quadruped.

11. The grain of any one of claims 1 to 10, wherein the coldblooded animal is selected from one or more of: a) a fresh water animal; b) a salt water animal; and c) a fish, arthropod, reptile and amphibian.

12. The grain of any one of claims 1 to 1 1 , wherein the grain is a cereal grain or a legume grain.

13. The grain of any one of claims 1 to 12, wherein the cereal grain is selected from: rice, barley, wheat, corn, erinkorn, farro, freekeh, horasan, millet, oats, quinoa, rye, sorghum, spelt, teff and buckwheat.

14. The grain of claim 13, wherein the rice is Oryza sativa.

15. The grain of any one of claims 1 to 14, wherein when the grain is a cereal grain the outer layer is the bran layer.

16. The grain of any one of claims 1 to 15, wherein when the grain is a cereal grain the inner layer is the endosperm.

17. The grain of any one of claims 1 to 12, wherein the grain is a pulse from a legume.

18. The grain of claim 17, wherein the pulse is selected from: bean, lentil, pea, chickpea and soybean.

19. The grain of any one of claims 1 to 1 1 , 17 or 18, wherein when the grain is a pulse from a legume the outer layer is the seed coat or the testa.

20. The grain of any one of claims 1 to 1 1 or 17 to 19, wherein when the grain is a pulse from a legume the inner layer is the cotyledon.21 . The grain of any one of claims 1 to 20, wherein at least one fracture has a width of about 50 pm to about 500 pm, or about 100 pm to about 450 pm, or about 150 pm to about 400 pm, or about 200 pm to about 350 pm, or about 250 pm to about 300 pm.

22. The grain of any one of claims 1 to 21 , wherein at least one fracture has a length of about 50 pm to about 500 pm, or about 100 pm to about 450 pm, or about 150 pm to about 400 pm, or about 200 pm to about 350 pm, or about 250 pm to about 300 pm.

23. The grain of any one of claims 1 to 22, wherein at least one fracture spans the depth of the outer layer.

24. The grain of any one of claims 1 to 23, wherein at least one fracture provides an opening to the inner layer.

25. A method for preparing a scaffold for culturing animal cells comprising forming at least one fracture in the outer layer of a grain.

26. The method of claim 25, wherein at least one fracture spans the depth of the outer layer.

27. The method of claim 25 or claim 26, wherein at least one fraction provides an opening to the inner layer.

28. The method of any one of claims 25 to 27, wherein the grain is a cereal grain, and the outer layer is the bran layer and the inner layer is an endosperm.

29. The method of any one of claims 25 to 27, wherein the grain is a pulse from a legume, the outer layer is the seed coat, or the testa and the inner layer is the cotyledon.

30. The method of any one of claims 25 to 29, wherein the method reduces the net mass of the grain.

31. The method of any one of claims 25 to 30, wherein the method comprises removing starch and / or arsenic from the grain.

32. The method of any one of claims 25 to 31 , wherein the method comprises: a) detoxifying the grain; b) sterilizing the grain; and c) washing the sterilised grain.

33. The method of claim 32, wherein sterilising comprises autoclaving, alcohol based sterilization, or UV-light sterilization.

34. The method of claim 32 and claim 33, wherein washing the sterilised grain comprises washing in phosphate buffered saline (PBS).

35. The method of any one of claims 32 to 34, wherein the method comprises treating the grain with one or more of: a) low pH; b) high pH; c) sonication; d) high salt; e) low temperature; f) high temperature; g) physical disruption; and h) two or more of a) to g).

36. A scaffold for culturing animal cells comprising a grain having an outer layer with at least one fracture in the outer layer of the grain.

37. The scaffold of claim 26, wherein the scaffold is edible.

38. The scaffold of claim 36 or claim 37, produced by or obtainable by the method of any one of claims 25 to 35.

39. A method of producing a cell culture product, the method comprising: a) adding the scaffold of any one of claims 36 to 38 and one or more animal cells to a cell culture apparatus; andb) culturing the cells.

40. The method of claim 39, wherein the cell culture apparatus is a bioreactor.41 . The method of claim 40, wherein the bioreactor is a stirred bioreactor.

42. A cell culture product comprising the grain of any one of claims 1 to 24, or the scaffold of any one of claims 36 to 38.

43. The cell culture product of claim 42, wherein the product is a meat substitute.

44. The cell culture product of claim 42 or claim 43, wherein the product is a cultured meat product.

45. The cell culture product of any one of claims 42 to 44, wherein the cell culture product mimics a property of a traditional slaughtered meat.

46. The cell culture product of any one of claims 42 to 45, wherein the product is not genetically modified.

47. The cell culture product of any one of claims 42 to 46, wherein the product is cooked or dehydrated.

48. A grain comprising: i) an outer bran layer with at least one fracture spanning the bran layer; ii) an inner endosperm; and iii) at least one animal cell between the bran layer and the endosperm and / or at least partially in the fracture.

49. A milled polished grain comprising a fracture in the outer layer of the grain and at least one cell at least partially in the fracture.

50. A scaffold for culturing animal cells comprising a milled polished grain comprising a fracture in the outer layer of the grain and at least one cell at least partially in the fracture. 51 . A method for preparing a scaffold for culturing animal cells comprising forming a fracture in the outer layer of a milled polished grain.

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