Bioreactor, method and system for producing cultivated meat

Abstract

The present disclosure related to a packed bed bioreactor, methods for using the same, cultured cell mass and food products comprising the cultured cell mass.

Description

[0001] BIOREACTOR, METHOD AND SYSTEM FOR PRODUCING CULTIVATED MEAT

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to the production of cultivated meat.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] International application publication No. W02022038240

[0007] International application publication No. WO2022097139

[0008] International application publication No. W02012140519

[0009] US application publication No. US2021189329

[0010] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0011] BACKGROUND

[0012] WO20220382 describes edible hollow fibers and cartridges and bioreactors comprising the hollow fibers that comprise one or more materials selected from the group consisting of hydrocolloids and proteins, having an outer diameter of about 0.2 mm to about 2.0 mm, a porosity of 0% to about 75% and a wall thickness of about 0.05 mm to about 0.4 mm. Also described by WO20220382 are methods of production of structured clean meat products produced with the hollow fibers, cartridges and bioreactors and the structured clean meat products produced by the methods.

[0013] WO2022097139 describes the large-scale production of cultured cells, and involves systems that comprise a plurality of scaffolds arranged optionally in a multi-layer configuration. W02022097139 also describes methods of use of the systems for production of cells and / or tissue cultures for a variety of uses, including the production of cultured food products, particularly cultured meat.

[0014] W02012140519 describes of using vibration to harvest cells grown in 3D culture. The methods entail the application of force to cells attached to a 3D matrix of sufficient amplitude, frequency, and duration to detach cells from the matrix and to flush the detached cells out of the matrix material. An apparatus for performing the disclosed methods.

[0015] US2021189329 describes methods and systems for enhanced production and / or secretion of extracellular vesicles from at least one three-dimensional porous scaffold having a population of stem cells cultured thereon, utilizing various shear stress conditions on a variety of stem cells.

[0016] GENERAL DESCRIPTION

[0017] The presently disclosed subject matter is based on the finding that cells and tissue can be effectively grown on edible scaffold within a packed bed bioreactor, to thereby result in an edible mass that can form into an alternative food product.

[0018] Thus, in accordance with a first aspect of the presently disclosed subject matter, there is provided a packed bed bioreactor comprising a basin having a first end, a second end and a wall extending between said first end and said second end defining an internal space, said bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein said internal space is filled with plurality of porous three-dimensional scaffold structures; wherein said plurality of porous three-dimensional scaffold structures comprises edible material; wherein said three-dimensional scaffold structures are packed within said basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of said porous three-dimensional scaffold structures within said internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet.

[0019] In accordance with a second aspect of the presently disclosed subject matter there is provided a method comprising:

[0020] (i) introducing cell media comprising cells into a packed bed bioreactor comprising a basin having a first end, a second end and a wall extending between said first end and said second end defining an internal space, said bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein said internal space is filled with plurality of porous three- dimensional scaffold structures; wherein said plurality of porous three-dimensional scaffold structures comprises edible material; wherein said three-dimensional scaffold structures are packed within said basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of said porous three-dimensional scaffold structures within said internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet;

[0021] (ii) providing conditions that support growth and / or differentiation of said cells on said porous three-dimensional scaffold structures; and

[0022] (iii) harvesting said porous three-dimensional scaffold structures with cells thereon.

[0023] In accordance with a third aspect of the presently disclosed subject matter, there is provided an edible cultured cell mass comprising plurality of porous three-dimensional scaffold structures that includes edible material and at least cells carried (adsorbed on, embedded within the pores etc.) by said edible material. In accordance with a fourth of its aspects of the presently disclosed subject matter, there is provided a food product comprising an edible cultured cell mass comprising plurality of porous three-dimensional scaffold structures that includes edible material and at least cells carried by the said edible material; said three-dimensional scaffold structures being distributed within said mass such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure.

[0024] EMBODIMENTS

[0025] Some embodiments of this disclosure will now be described in the following numbered paragraph. The following description intends to add on the above general description and not limit it in any manner.

[0026] 1. A packed bed bioreactor comprising a basin having a first end, a second end and a wall extending between said first end and said second end defining an internal space, said bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein said internal space is filled with plurality of porous three-dimensional scaffold structures; wherein said plurality of porous three-dimensional scaffold structures comprises edible material; wherein said three-dimensional scaffold structures are packed within said basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of said porous three-dimensional scaffold structures within said internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet.

[0027] 2. A method comprising:

[0028] (i) introducing cell media comprising cells into a packed bed bioreactor comprising a basin having a first end, a second end and a wall extending between said first end and said second end defining an internal space, said bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein said internal space is filled with plurality of porous three- dimensional scaffold structures; wherein said plurality of porous three-dimensional scaffold structures comprises edible material; wherein said three-dimensional scaffold structures are packed within said basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of said porous three-dimensional scaffold structures within said internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet;

[0029] (ii) providing conditions that support growth of said cells on said porous three- dimensional scaffold structures; and

[0030] (iii) harvesting said porous three-dimensional scaffold structures with cells thereon.

[0031] 3. The packed bed bioreactor and / or method of Embodiment No.1 or 2, wherein said edible material comprises plant material.

[0032] 4. The packed bed bioreactor and / or method of any one of Embodiment No.l to 3, wherein said edible material comprises at least insoluble dietary fibers.

[0033] 5. The packed bed bioreactor and / or method of Embodiment No. 4, wherein said insoluble dietary fibers comprise polysaccharides.

[0034] 6. The packed bed bioreactor and / or method of any one of Embodiments No.l to 5, wherein said edible material comprises cellulose material.

[0035] 7. The packed bed bioreactor and / or method of any one of Embodiments No.1 to 6, wherein said edible material comprises a plant protein.

[0036] 8. The packed bed bioreactor and / or method of Embodiment No. 3, wherein said edible material is a fruit or vegetable derived material.

[0037] 9. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 8, wherein said edible material comprises at least insoluble dietary fibers derived from fruits. 10. The packed bed bioreactor and / or method of Embodiment No. 1, wherein said edible material comprises animal-derived material.

[0038] 11. The packed bed bioreactor and / or method of Embodiment No. 10, wherein said animal-derived material is selected from the group consisting of decellularized tissue, collagen, gelatin, elastin, laminin, fibronectin, vitronectin and any combination of same.

[0039] 12. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 11 , wherein said edible material comprises a combination of plant material and animal-derived material.

[0040] 13. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 12, wherein each of said porous three-dimensional scaffold structures comprise at least one dimension having a size within a range of about 0.5mm and about 500mm.

[0041] 14. The packed bed bioreactor and / or method of Embodiment No. 13, wherein said porous three-dimensional scaffold structures has a first dimension and a second defining together a plane, and a thickness perpendicular to said plane, said thickness being within a range of about 0.1mm and about 2mm.

[0042] 15. The packed bed bioreactor and / or method of Embodiment No. 14, wherein at least one of said first dimension and said second dimension is greater than said thickness.

[0043] 16. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 15, wherein the porous scaffold comprises a porous material comprises pores having a size of between about 10pm and about 800pm,

[0044] 17. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 16, wherein said plurality of porous three-dimensional scaffold structures have a porosity ranging from between about 50% and about 99%.

[0045] 18. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 17, wherein said plurality of porous three-dimensional scaffold structures are packed with a packing dry density ranging from between about 1 mg / ml and lOOOmg / ml. 19. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 18, wherein said plurality of porous three-dimensional scaffold structures are packed with a packing wet density ranging from between about 0.1 g / ml and about 1.0 g / ml.

[0046] 20. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 19, wherein said three-dimensional scaffold structures have a shape selected from the group consisting of sheets, fibers, beads, flaks, discs, spheres, cylinders, rings, polygons and star shapes.

[0047] 21. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 20, comprising at least one type of three-dimensional scaffold structures.

[0048] 22. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 20, comprising at least two different types of three-dimensional scaffold structures.

[0049] 23. The packed bed bioreactor and / or method of Embodiment 22, wherein said at least two types of three-dimensional scaffold structures are different in at least one of scaffold material, scaffold size, scaffold shape, scaffold stiffness, scaffold elasticity, scaffold average pore size, scaffold porosity, scaffold color, scaffold texture or a combination thereof.

[0050] 24. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 23, wherein said plurality of scaffold structures is arranged in a homogenous mixture distribution.

[0051] 25. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 24, wherein said plurality of scaffold structures is arranged in a non-homogenous organized distribution.

[0052] 26. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 25, wherein said porous three-dimensional scaffold structures are pre-conditioned to promote adherence of cells onto said scaffold structures.

[0053] 27. The packed bed bioreactor and / or method of Embodiment No. 26, wherein said preconditioning comprises physical and / or chemical change of surface of said porous three- dimensional scaffold structures. 28. The packed bed bioreactor and / or method of Embodiment No. 26 or 27, wherein said pre-conditioning comprises any one of plasma treatment, electrostatic charging of surface of said porous three-dimensional scaffold structures; increasing surface energy of said porous three-dimensional scaffold structures, removing lignin from surface of said porous three-dimensional scaffold structures; exposing cellulose residues on surface of said porous three-dimensional scaffold structures.

[0054] 29. The packed bed bioreactor and / or method of any one of Embodiments No. 28 to 27, wherein said preconditioning is determined by level of adherence of cells onto said porous three-dimensional scaffold structures under conditions comprising positive charge and exposed cellulose binding sites.

[0055] 30. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 29, wherein said porous three-dimensional scaffold structures within said basin are lyophilized structures.

[0056] 31. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 30, wherein said porous three-dimensional scaffold structures are sterilized.

[0057] 32. The packed bed bioreactor and / or method of Embodiment No. 32, wherein said porous three-dimensional scaffold structures are sterilized by any sterilization method selected from autoclaving, UV irradiation, radiation sterilization and dry heat sterilization.

[0058] 33. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 32, comprising a removable headplate configured to allow introduction and / or removal of content of said basin.

[0059] 34. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 3332, comprising at least one port for accommodating at least one sensor.

[0060] 35. The packed bed bioreactor and / or method of Embodiment No. 34, wherein said at least one sensor is selected to detect at least one parameter selected from the group consisting of pH, dissolved oxygen (DO), temperature capacitance, glucose, lactate, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH), biomass weight, biomass level in basin, foam. 36. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 35, comprising a mechanism configured to allow fluid circulation within said internal space.

[0061] 37. The packed bed bioreactor and / or method of Embodiment No. 36, wherein said mechanism comprises at least one component selected from the group consisting of impeller, peristaltic pump, diaphragm pump, vibrator, tilter and lifter.

[0062] 38. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 37, comprising at least one control loop.

[0063] 39. The packed bed bioreactor and / or method of Embodiment No. 38, wherein said at least one control loop is configured to maintain at least one parameter selected from the group consisting of pH, dissolved oxygen and temperature at a predetermined setpoint value.

[0064] 40. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 39, wherein said basin is positioned in an orientation to allow flow up of fluids from said fluid inlet upwards, and through the packed bed.

[0065] 41. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 40, wherein said basin comprises at least one transparent segment in said wall, configured to allow viewing content of said internal space.

[0066] 42. The packed bed bioreactor and / or method of any one of Embodiments No. 1 to 42, for use with a source of culture media for growing cells within said basin.

[0067] 43. The method of any one of Embodiments No. 1 to 42, comprising pre-conditioning said porous three-dimensional scaffold structures to promote adherence of cells onto said scaffold structures.

[0068] 44. The method of Embodiment No. 43, wherein said pre-conditioning comprises physical and / or chemical change of surface of said porous three-dimensional scaffold structures.

[0069] 45. The method of Embodiment No. 43 or 44, wherein said pre-conditioning comprises any one of plasma treatment, electrostatic charging of surface of said porous three- dimensional scaffold structures; increasing surface energy of said porous three- dimensional scaffold structures, removing lignin from surface of said porous three- dimensional scaffold structures; exposing cellulose residues on surface of said porous three-dimensional scaffold structures.

[0070] 46. The method of any one of Embodiments No. 43 to 45, wherein said preconditioning is determined by level of adherence of cells onto said porous three-dimensional scaffold structures under conditions comprising positive charge and exposed cellulose binding sites.

[0071] 47. The method of any one of Embodiments No. 43 to 46, comprising lyophilization of said porous three-dimensional scaffold structures prior to contacting said structures with said cells.

[0072] 48. The method of any one of Embodiments No. 43 to 47, comprising sterilization of said porous three-dimensional scaffold structures prior to contacting said structures with said cells.

[0073] 49. The method of Embodiment No. 48, wherein said sterilization comprises any sterilization method selected from the group consisting of autoclaving, UV irradiation, radiation sterilization and dry heat sterilization and combination of same.

[0074] 50. The method of any one of Embodiments No. 43 to 49, comprising washing said porous three-dimensional scaffold structures with an solution, said washing is subsequent to said pre-conditioning, if said pre-conditioning is performed.

[0075] 51. The method of Embodiment No. 50, wherein said solution is selected from the group consisting of PBS, fibronectin attachment solution, collagen attachment solution, laminin attachment solution, vitronectin attachment solution, elastin attachment solution poly-L-lysine attachment solution, gelatin attachment solution and spent attachment solution.

[0076] 52. The method of any one of Embodiments No. 43 to 51 , comprising saturating said porous three-dimensional scaffold structures with a growth media, prior to introducing said cell media and subsequent to said washing.

[0077] 53. The method of any one of Embodiments No. 43 to 52, wherein said cell media comprises a single cell type or more than one cell type.

[0078] 54. The method of any one of Embodiments No. 43 to 53, wherein said cell media comprises at least stem cells. 55. The method of any one of Embodiments No. 43 to 54, wherein said cell media comprises at least cells selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, adult stem cells, mesenchymal stem cells, myoblasts, satellite muscle cells, fibroblasts, hepatoblasts, osteoblasts, chondroblasts, adipocytes, hepatocytes, osteocytes, chondrocytes, and any combination of same.

[0079] 56. The method of any one of Embodiments No. 43 to 55, wherein said conditions that support growth of said cells on said porous three-dimensional scaffold structures comprise at least one condition selected from the group consisting of temperature, pH, dissolved oxygen (DO) and agitation.

[0080] 57. The method of any one of Embodiments No. 43 to 56, wherein said conditions that support growth of said cells on said porous three-dimensional scaffold structures comprise temperature within a range of between about 20°C and about 45°C.

[0081] 58. The method of any one of Embodiments No. 43 to 57, wherein said conditions that support growth of said cells on said porous three-dimensional scaffold structures comprise pH within a range of between about 6 and about 8.

[0082] 59. The method of any one of Embodiments No. 43 to 58, wherein said conditions that support growth of said cells on said porous three-dimensional scaffold structures comprise level of DO within a range of between about 10% and about 100%.

[0083] 60. The method of any one of Embodiments No. 43 to 59, wherein said conditions that support growth of said cells on said porous three-dimensional scaffold structures comprise agitation with a shear stress of between about O.ldyn / cm2and about 10dyn / cm2.

[0084] 61. The method of any one of Embodiments No. 43 to 60, wherein said conditions that support growth of said cells on said porous three-dimensional scaffold structure comprise conditions supportive of cell seeding and conditions supporting of cell growth.

[0085] 62. The method of Embodiment No. 61, wherein said conditions comprise flow velocity between about 1 cm / sec and 5cm / sec.

[0086] 63. The method of Embodiment No. 62, wherein agitation during said seeding comprises agitation with alternating velocities including a first rotational speed in a range of 10-50rpm and a second rotational speed between lOOrpm and 200rpm. 64. The method of Embodiment No. 63, wherein said agitation at said first rotational speed is for a period of between 10 minutes and 20 minutes, and said agitation at said second rotational speed is for a period of less than 1 minute.

[0087] 65. The method of any one of Embodiments No. 63 to 64, wherein said agitation during said cell growth is at a rotational speed within a range of about lOOrpm and about 200rpm.

[0088] 66. The method of any one of Embodiments No. 43 to 65, comprising monitoring at least one parameter selected from the group consisting of pH, dissolved oxygen (DO), temperature capacitance, metabolites, biomass weight, biomass level in basin and foam.

[0089] 67. The method of Embodiment No. 66, wherein said metabolites comprises at least one metabolite selected from the group consisting of glucose, lactate, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH).

[0090] 68. The method of any one of Embodiments No. 43 to 67, perfusing growth and / or differentiation media through said basin.

[0091] 69. The method of any one of Embodiments No. 43 to 68, comprising providing said conditions that support growth of said cells.

[0092] 70. The method of any one of Embodiments No. 43 to 69, comprising providing said conditions that support differentiation of at least part of said cells.

[0093] 71. The method of any one of Embodiments No. 43 to 70, wherein said cells are undifferentiated cells and harvesting takes place when glucose consumption rate reaches a stationary phase.

[0094] 72. The method of Embodiment No. 71 , wherein said cells comprise differentiated cells and harvesting takes place between 1 to 30 days after initiating perfusion with differentiation media.

[0095] 73. The method of any one of Embodiments No. 43 to 72, comprising collecting harvested porous three-dimensional scaffold structures with at least cells grown thereon.

[0096] 74. The method of any one of Embodiments No. 43 to 73, comprising collecting harvested porous three-dimensional scaffold structures with cell tissue grown thereon. 75. The method of Embodiment No. 73 or 74, wherein at least part of the cells is embedded within pores of the porous structures.

[0097] 76. An edible cultured cell mass comprising plurality of porous three-dimensional scaffold structures that includes edible material and at least cells carried by said edible material, said three-dimensional scaffold structures being distributed within said mass such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure.

[0098] 77. The edible cultured cell mass of Embodiment No. 76, wherein said plurality of three-dimensional scaffold structures comprise, adsorbed thereon, any one or combination of undifferentiated cells, differentiated cells, extracellular matrix protein and cell secreted metabolites.

[0099] 78. The edible cultured cell mass of Embodiment No. 76 or 77, being characterized by at least one of the following: protein content of between about 0.5-20% wet weight out of a total wet weight of said mass; lipid content of between about 0.1-40% wet weight out of a total wet weight of said mass; lipid to protein weight ratio of between about 0.1-1; collagen to protein weight ratio of between about 1-50%.

[0100] 79. A food product comprising an edible cultured cell mass comprising plurality of porous three-dimensional scaffold structures that includes edible material and at least cells carried on said edible material; said three-dimensional scaffold structures being distributed within said mass such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure.

[0101] 80. The food product of Embodiment No. 78, wherein said cultured cell mass is as defined in any one of Embodiments No. 76 to 78.

[0102] 81. A cultivating system comprising - a packed bed bioreactor comprising a container having a first end, a second end and a wall extending between said first end and said second end defining an internal space, said bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein said internal space is filled with plurality of porous three-dimensional scaffold structures; wherein said plurality of porous three-dimensional scaffold structures comprises edible material, wherein said three-dimensional scaffold structures are packed within said basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of said porous three-dimensional scaffold structures within said internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet;

[0103] - a cell media reservoir in fluid communication with said internal space;

[0104] - an oxygen source in fluid communication with said internal space;

[0105] - a control module configured for controlling at least one parameter during operation of said bioreactor.

[0106] 82. The cultivating system of Embodiment No. 81 , wherein said basin is positioned in an orientation to allow flow up of fluids from said first fluid inlet upwards, and through the packed bed.

[0107] 83. The cultivating system of Embodiment No. 81 or 82 wherein said fluid outlet is located on said second end.

[0108] 84. The cultivating system of any one of Embodiments No. 81 to 83, comprising at least one port accommodating at least one sensor, and said control module is configured to receive data from said at least one sensor and operate said bioreactor based on said received data. 85. The cultivating system of any one of Embodiments No. 81 to 84, comprising at least one pump for directing flow of fluid from said cell media source and / or oxygen source into said internal space.

[0109] 86. The cultivating system of any one of Embodiments No. 81 to 85, wherein said packed bed bioreactor is as defined in any one of Embodiment No. 1 to 42.

[0110] 87. The cultivating system of any one of Embodiments No. 81 to 85, configured for performing the method of any one of Embodiments No. 1 to 75.

[0111] BRIEF DESCRIPTION OF THE DRAWINGS

[0112] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0113] Figures 1A-1C are schematic illustrations of exemplary edible packed bed (EPB) bioreactor configurations, Figure 1A an EPB having homogeneous distribution of a single type of edible scaffold structures, Figure IB homogenous mixture of two different edible scaffold structures and Figure 1C an organized yet separated distribution of two different edible scaffold structures.

[0114] Figures 2A-2I show the effect of different edible scaffold materials on cell growth, Figure 2A is a bar graph showing fold increase of poultry fibroblast cells or poultry adult stem cells (ASCs) growth on different scaffold materials at different days, scaffold material include fungi scaffold, Textured Soy Protein (TSP) scaffold; Textured Vegetable Protein (TVP) scaffold, decellularized (DC) plant stem scaffold, DC-vegetable root scaffold, DC- fruit scaffold (poultry fibroblast cells), and Gelatex type 1 scaffold and Gelatex type 2 scaffold (ASCs), Figures 2B-2I are confocal microscopy images of cell grown on scaffold materials fungi scaffold, Textured pea Protein (TPP) scaffold; TVP scaffold, DC plant stem scaffold, DC-vegetable root scaffold, DC-fruit scaffold, Gelatex type 1 scaffold and Gelatex type 2 scaffold, cells were stained for Dapi (4',6-diamidino-2-phenylindole, blue) and F-actin (green); scale bar 100 pm.

[0115] Figures 3A-3C show growth of poultry ASC on edible scaffolds with different geometry and size, Figure 3A is a bar graph showing thickness measures of scaffolds with different geometry with different views (Top and side) of the scaffolds, Hex - hexagonal scaffold and Disc - circular shape scaffold, Figure 3B is a graph showing glucose consumption rate (GCR) of cells grown on different scaffolds in EPB bioreactor, Figure 3C is a graph showing soluble protein from cells grown on different scaffolds, soluble protein was determined by bicinchoninic acid (BCA) assay.

[0116] Figures 4A-4D show the effect of plasma treatment on cell attachment and growth in edible scaffolds; Figures 4A and 4B are confocal images of stained cellulose (CBD- GFP) of untreated decellularized plant scaffold and of plasma-treated decellularized plant scaffold, respectively, Figure 4C is a graph showing cell growth on plasma treated and untreated scaffolds, Figure 4D is a graph showing soluble protein in plasma treated and untreated scaffolds.

[0117] Figures 5A-5D show the effect of different solutions on cell attachment and growth in edible scaffolds; Figure 5A is a graph showing cell growth on scaffold treated with fibronectin factor attachment solution (“with attachment”), waste media of cultured cells (“spent medium”), and PBS (“W / O attachment”), at different days, Figures 5B-5D are confocal images of cells seeded on edible scaffold washed with solutions, Figure 5B scaffold washed with fibronectin factor attachment solution, Figure 5C scaffold washed with waste media of cultured cells and Figure 5D scaffold washed with PBS; cells stained for Dapi (blue) and F-actin (green), scale bar 1mm.

[0118] Figures 6A-6H are confocal microscopy images of different cell types grown on scaffolds in EPB bioreactor; Figures 6A and 6E show poultry ASCs, Figures 6B and 6F show poultry fibroblast cells, Figures 6C and 6G show bovine ASCs, and Figures 6D and 6H show bovine fibroblast cells, Figures 6A-6D scale bar of 1000 pm and Figures 6E- 6H a scale bar of 100 pm, cells stained for Dapi (blue) and F-actin (green).

[0119] Figures 7A-7N show cell growth distribution on scaffold and in between scaffolds in EPB bioreactor; Figures 7A and 7B are confocal microscopy images of cell’s nucleus (stained with Dapi) and actin filaments (stained with Phalloidin) in cells located in opposite sides of a scaffold, Figures 7C and 7D are images of the clusters of scaffolds with cells cultured in between the scaffold forming biomass as harvested from bioreactor, Figures 7E-7N are images showing cell distribution at different depths of a scaffold (cells stained using Sirius-red), scaffold 1 and scaffold 2 are repetitions of the same type of scaffold.

[0120] Figures 8A-8D are graphs showing monitoring of metabolites during cultivated cell (meat) grown in an EPB bioreactor, Figure 8A glucose consumption rate, Figure 8B NH3 formation rate, Figure 8C lactate formation rate, and Figure 8D lactate dehydrogenase (LDH) rate.

[0121] Figures 9A-9H show characteristics of tissue formation and nutrient content on edible scaffold in a EPB bioreactor; Figure 9A cells stained with Dapi and F-actin indicative of cell growth, Figure 9B cells stained with collagen indicative of Extra Cellular Matrix (ECM), Figure 9C cells stained with Oil Red O indicative of triglyceride droplets, Figures 9D-9F are graphs showing protein content of cells grown in EBP bioreactor, as control cells were grown in Erlenmeyer or as cell pellet, and Figures 9G-9H show triglyceride (TG) content indicating cell differentiation into fat cells.

[0122] Figures 10A-10D are graphs showing the distribution of cell cultivated on edible scaffolds; Figure 10A is a graph showing analysis of viable cells cultivated on the edible scaffold structures in the packed bed bioreactor from "Top" or "Bottom" areas of the packed bed and compared to two control groups of cells (with scaffold) grown in well plate according to a "Direct" and a "Suspension" technique, Figure 10B is a graph showing distribution of cells cultivated on edible scaffolds in a large packed bed bioreactor in different areas of the packed bed represented by the red dot (different floors of the packed- bed, different height within the packed bed). Figure IOC is a graph showing number of cells in different floors of the packed-bed and in a smaller bioreactor, Figure 10D is a graph showing number of cells at different proximity to the center of a large packed bed compared to small biotrector.

[0123] Figures 11A-11D show differentiation of cells into matured muscle cells in EPB bioreactor; Figures 11A and 11B are confocal images showing formation of matured muscle fibers on edible scaffold indicated by nuclei staining (DAPI- Blue) and Myosin Heavy Chain (MyHC - Red) staining at different magnifications, Figure 11C is an image of a western blot showing presence of MyHC, band at molecular weight of 240 kDa, and Figure 11D is a graph showing quantitative analysis of MyHC from the western blot of Figure 11C. Figures 12A-12C are images showing Figure 12A a packed bed bioreactor, Figure 12B cell grown in the packed bed bioreactor, Figure 12C cooked cultivated tissue (cultivated meat).

[0124] Figure 13 is a bar graph showing average score of chicken smell intensity ranking in smelling test.

[0125] DETAILED DESCRIPTION

[0126] The presently disclosed subject matter is based on the finding that cells and tissue can effectively grow and differentiate on edible scaffold within a packed bed bioreactor, to provide an edible tissue mass that can form into an alternative food product, including, inter alia, alternative meat product or alternative fish and sea food products.

[0127] As shown herein, the edible scaffolds were designed to support growth of cells both on their entire surfaces (along the perimeter) and within their porous structures (in the scaffold pores / voids / lumen), thus creating a three-dimensional environment that closely mimics natural tissue. Specifically, Figures 7 A and 7B show growth of cells on two opposite sides of an exemplary scaffold, Figures 7C and 7D show growth of cells between scaffolds and Figures 7E-7N show growth of cells at various depths within the scaffold. These results demonstrate the unique features of the scaffold allowing uniform cell distribution of cells on the scaffold and within the scaffold pores This suggests that the scaffold can support cell attachment, cell growth and proliferation as well as cell differentiation not only on the scaffold surface but also within the pores and voids of the scaffold structure, forming a continuous 3-dimentional (3D) structure allowing formation of connected tissue.

[0128] As described herein, the plurality of the edible scaffolds is packed within the bioreactor in a unique close proximity configuration (arrangement) such that at least part of a surface of one scaffold is in contact with at least part of a surface of a neighboring scaffold, essentially without forming spaces / voids between the scaffolds. This close proximity arrangement allows uniform cell distribution and nutrient delivery.

[0129] As also described herein, the packed bed bioreactor configuration enables flow of both liquid and gas in a manner that surround the scaffold structure, thereby immersing (wrapping) the scaffold and the cells seeded on the scaffold in nutrients and gas. This design ensures that the nutrients and gas are in close proximity to the cells, allowing for the effective delivery of these essential components and overcoming diffusion limited delivery. By eliminating diffusion constraints, the bioreactor configuration ensures that fresh nutrients and oxygen are consistently available to all cells, while metabolic waste products efficiently removed.

[0130] It was suggested that despite of the fact that the scaffolds are packed in close proximity (without essentially forming voids / spaces), the bioreactor enabled a uniform flow throughout, ensuring a homogenous and continuous distribution of nutrients and gases as well as removal of cell byproducts, to overall support cell growth and differentiation. As shown in Figure 8, an increase in glucose consumption rate, ammonia formation rate, lactate formation rate and lactate dehydrogenase formation rate were observed with time suggesting growth (proliferation) of cells on the edible scaffolds. In addition, as shown in Figure 9, an increase in collagen production was observed, suggesting formation of tissue. Furthermore, as shown in these figures, the cells underwent differentiation into fat cells as demonstrated by the presence of triglyceride. In addition, as shown in Figure 11 , cells were capable of differentiating into muscle cells forming muscle tissue.

[0131] Interestingly, the protein concentration and the triglyceride concentration were higher in cells grown on edible scaffold within the packed bed as compared to cells grown on edible scaffold in suspension (in an Erlenmeyer).

[0132] As also shown in Figure 10, the growth and of the cells was not affected from the position of the scaffolds within the bioreactor, suggesting that the bioreactor supports homogenous growth of the cells.

[0133] Without being bound by theory, it was suggested that the ability of the scaffold to support cell growth and differentiation on and within the scaffold together with the close proximity configuration between the plurality of scaffolds within the bioreactor, facilitated interactions between cells grown on neighboring scaffolds, including, inter alia, cell-cell signaling, cell motility, nutrient transport or cell-cell adhesion, forming a three- dimensional network of interconnected cells resulting in tissue formation. Thus, in accordance with a first aspect of the present disclosure, there is provided a packed bed bioreactor comprising a basin having a first end, a second end and a wall extending between the first end and the second end defining an internal space, the bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein the internal space is filled with plurality of porous three-dimensional scaffold structures; wherein the plurality of porous three-dimensional scaffold structures comprises edible material; wherein the three-dimensional scaffold structures are packed within the basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of the porous three-dimensional scaffold structures within the internal space are selected to allow essentially uniform flow of fluid from the first fluid inlet to the fluid outlet.

[0134] In the following disclosure, when referring to a packed bed bioreactor it is to be understood as referring to method, edible scaffold structure, edible cultured cell mass, food products and cultivating systems. Thus, whenever providing a feature with reference to the packed bed bioreactor it is to be understood as defining the same feature with respect to the method, edible scaffold structure edible cultured cell mass, food products and cultivating systems, mutatis mutandis.

[0135] The packed bed bioreactor as used herein refers to a bioreactor in which growth of cells occurs on solid supports. The solid support is or comprises the scaffold as described herein.

[0136] Plurality of porous three-dimensional scaffold structures as used herein encompasses multiple scaffolds of the same type or of different types as further described.

[0137] The term scaffold as used herein refers to a three-dimensional structure including a material that is suitable for adherence of cells thereto and for further growth and / or differentiation of the cells. The scaffold in accordance with the present disclosure is characterized by mechanical properties (e.g. stiffness, elasticity) that supports cell attachment, proliferation, and differentiation into tissue such as meat or fish. It was suggested that this unique mechanical property of the scaffold along with the unique distribution of the scaffolds within the bioreactor, closely resemble the natural extracellular matrix environment.

[0138] Hence in accordance with some embodiments, the scaffold is characterized by an elastic modulus of between about 3kPa and about 600 kPa, at times between about 3kPa and about 100 kPa, t times between about lOOkPa and about 600 kPa.

[0139] The term elastic modulus also known as modulus of elasticity, refers to a measure of a material's (e.g. scaffold) stiffness or rigidity that quantifies the relationship between stress (force per unit area) and strain (proportional deformation) in a material subjected to elastic deformation. The elastic modulus is defined as the ratio of stress to strain within the range where the material returns to its original shape after the stress is removed.

[0140] The elastic modulus can be measured experimentally through various methods known in the art, depending on the type of material and the specific modulus being measured (e.g., Young's modulus, shear modulus, or bulk modulus).

[0141] In accordance with some embodiments, the scaffold is characterized by a stiffness of between about 3kPa and about 15kPa.

[0142] The term stiffness as used herein refers to a measure of a material's (e.g. scaffold) resistance to deformation under an applied load and provides an indication of how much force is required to achieve a certain amount of deformation.

[0143] The stiffness can be measured experimentally through various methods known in the art.

[0144] Without being bound by theory, it was suggested that due to the unique arrangement of the scaffold being in close proximity to one another, the scaffold stiffness can be reduced to better accommodate cell growth and tissue formation

[0145] As described herein, the scaffold structure comprises an edible material. In some embodiments, the scaffold structure is entirely made from an edible material. In accordance with such embodiments, the entire scaffold is edible. The present application is not limited to a specific edible material and can be applicable to a variety of edible materials, for example such edible materials that are suitable for human or animal consumption.

[0146] As shown in Figures 2A-2I, the material of the scaffold is not limited to a specific type, as various materials including scaffold material derived from fungi, vegetable, and fruit as well as processed materials including TSP and TVP have been shown to support cell growth. These experiments have validated that multiple material, whether natural or synthetic, can effectively support the porous structure necessary for three-dimensional cell growth and cellular interactions.

[0147] In some examples, the edible material is not from an animal source.

[0148] In some examples, the edible material comprises a plant material, an algae material, a fungus material or any combination thereof.

[0149] In some examples, the edible material is or comprises a plant material.

[0150] The plant material as used herein refers to any substance derived from a plant, including any part thereof, or a plant source and encompasses one or more of seeds, leaves, stems, or roots.

[0151] The plant material may be in accordance with some examples, a decellularized (DC) plant tissue. The decellularization plant may be obtained by any method known in the art which removed the native plant tissue and maintaining decellularized scaffold.

[0152] In some examples, the plant material is derived from a legume, a grain, a vegetable, a fruit, a nut or a seed.

[0153] In some embodiments, the legume is chickpea, clover, beans, peas, lentils, lupins, mesquite, carob, soybeans, peanuts, tamarind or any combination thereof.

[0154] In some embodiments, the grain is wheat, rice, oat, or any combination thereof.

[0155] In some embodiments, the seed is derived canola seeds, sunflower seeds, rice seeds or any combination thereof.

[0156] In some examples, the edible material is a fruit derived material, a vegetable derived material or a combination thereof. In some examples, the edible material is or comprises a fruit derived material. In some examples, the fruit derived material is from an apple and / or a pear. In some examples, the apple is a red apple. In some examples, the apple is a green apple. In some examples, the fruit derived material is decellularized fruit derived material.

[0157] In some examples, the edible material is or comprises a vegetable derived material. In some examples, the vegetable derived material is from one or more of onion, carrot, broccoli, cucumber, potato, asparagus, cauliflower, peppers, parsley, bamboo stems or any combination thereof. In some examples, the onion is green onion. In some examples, the onion is white onion. In some examples, the onion is red onion. In some examples, the vegetable derived material is decellularized vegetable derived material.

[0158] In some examples, the edible material is or comprises edible leaves. In some examples, the edible material is one or more of spinach leaves, lettuce leaves, kale leaves or cabbage leaves. In some examples, the leaves are decellularized leaves.

[0159] In some examples, the edible material is or comprises edible stem.

[0160] In some examples, the edible material is or comprises edible root.

[0161] In some examples, the edible material is or comprises insoluble dietary fibers. In some examples, the insoluble dietary fiber is or comprises polysaccharides. In some embodiments, the polysaccharide is one or more of cellulose and hemicellulose.

[0162] In some examples, the edible material comprises cellulose material.

[0163] In some examples, the edible material is or comprises a plant protein.

[0164] In some examples, the plant protein is a legume protein. In some examples, the plant protein is a grain protein. In some examples, the plant protein is a vegetable protein. In some examples, the protein is a fruit protein.

[0165] In some examples, the edible material comprises at least insoluble dietary fibers derived from fruits.

[0166] The edible scaffold can include other materials, in addition to the plant-based materials. In some examples, the edible material comprises animal-derived material. In some examples, the animal-derived material is selected from the group consisting of decellularized tissue, collagen, gelatin, elastin, laminin, fibronectin, vitronectin and any combinations thereof.

[0167] In some examples, the edible material comprises a combination of plant material and animal-derived material.

[0168] The present disclosure is not limited to a specific porous scaffold structure and the porous scaffold can be of any type, any size or any shape provided that it allows growth and / or differentiation of cells. As shown herein below, the bioreactor is compatible with a variety of scaffolds.

[0169] As shown in Figures 3A-3C, scaffolds of varying shapes (including hexagonal shape and disc shape) and varying sizes (including 0.5mm and 1 mm) exhibited similar glucose consumption rates (Figure 3B) and protein levels (Figure 3C), indicating uniform cell activity irrespective of shape and size of the testes scaffolds.

[0170] Without being bound by theory, it was suggested that the bioreactor supports growth of cells on different scaffold regardless the scaffold’s physical dimensions (e.g. shape and size). Such physical dimensions do not affect the cellular activities of glucose consumption and protein synthesis.

[0171] In some examples, each of the porous three-dimensional scaffold structures comprise at least one dimension with a size within a range of between about 0.2mm and about 500mm, at times within a range of about 0.2mm and about 300mm, at times within a range of about 0.2mm and about 100mm, at times within a range of about 0.2mm and about 50mm, at times within a range of about 0.2mm and about 10mm, at times within a range of about 0.2mm and about 5mm, at times within a range of about 0.2mm and about 1mm.

[0172] In some examples, the at least one dimension of the porous three-dimensional scaffold structure has a size within a range of between about 0.2mm and about 300mm, at times between about 1mm and about 300mm, at times between about 1mm and about 200mm, at times between about 1mm and about 100mm, at times between about 1mm and about 50mm, at times between about 1mm and about 40mm, at times between about 1mm and about 30mm, at times between about 1mm and about 20mm, at times between about 1mm and about 10mm.

[0173] In some examples, each of the porous three-dimensional scaffold structures comprise at least one dimension have a size of about 0.1mm, at times about 0.2mm, at times about 0.3mm, at times about 0.4mm, at times about 0.5mm, at times about 0.6mm, at times about 0.7mm, at times about 0.8mm, at times about 0.9mm, at times about 1mm, at times about 2mm, at times about 3mm, at times about 4mm, at times about 5mm, at times about 6mm, at times about 7mm, at times about 8mm, at times about 9mm, at times about 10mm, at times about 15mm, at times about 20mm, at times about 25mm, at times about

[0174] 30mm, at times about 35mm, at times about 40mm, at times about 50mm, at times about

[0175] 60mm, at times about 70mm, at times about 80mm, at times about 90mm, at times about

[0176] 100mm, at times about 120mm, at times about 150mm, at times about 170mm, at times about 200mm, at times about 220mm, at times about 250mm, at times about 270mm, at times about 300mm, at times about 320mm, at times about 350mm, at times about 370mm, at times about 400mm, at times about 420mm, at times about 450mm, at times about 470mm, at times about 500mm. In some examples, each of the porous three-dimensional scaffold structures comprise at least one dimension have a size of about 0.5mm.

[0177] In some examples, the porous three-dimensional scaffold structures have a first dimension and a second dimension defining together a plane, and a thickness perpendicular to the plane.

[0178] In some examples, the first dimension and a second dimension have the same dimensions. In some examples, the first dimension and a second dimension have different dimensions.

[0179] In some examples, at least one of the first dimension and the second dimension is greater than the thickness. In some examples, at least one of the first dimension and the second dimension has the same dimension as the dimension of the thickness. In some examples, the first dimension and the second dimension have the same dimension as the thickness.

[0180] In some examples, the thickness is within a range of about 0.1mm and about 2mm.

[0181] In some examples, the thickness is between about 0.3mm and about 2mm, at times between about 0.5mm and about 2mm, at times between about 0.7mm and about 2mm, at times between about 0.9mm and about 2mm. In some examples, the thickness is within a range of about 0.1mm and about 1.7mm, at times between about 0.3mm and about 1.5mm, at times between about 0.5mm and about 1.5mm, at times between about 0.7mm and about 1.5mm.

[0182] In some examples, the thickness is at least about 0.1mm, at times at least about 0.2mm, at times at least about 0.3mm, at times at least about 0.5mm, at times at least about 0.7mm, at times at least about 1mm, at times at least about 1.2mm, at times at least about 1.5mm, at times at least about 1.7mm, at times at least about 2mm.

[0183] In some examples, the thickness is within a range of about 0.1mm and about 1mm, at times between about 0.1mm and about 0.9mm, at times between about 0.1mm and about 0.8mm, at times between about 0.2mm and about 0.9mm, at times between about 0.2mm and about 0.8mm.

[0184] In some examples, the thickness is about 0.1mm, at times about 0.3mm, at times about 0.5mm, at times about 0.7mm, at times about 1mm, at times about 1.2mm, at times about 1.5mm, at times about 1.7mm, at times about 2mm.

[0185] The scaffold in accordance with the present disclosure comprises a porous material featuring pores or voids that may be interconnected or non-interconnected.

[0186] In some examples, the pores of the scaffold are interconnected. In some examples, at least part of the pores of the scaffold are interconnected.

[0187] In some examples, the pores of the scaffold are non-interconnected.

[0188] As described herein, the porous structure of the scaffold facilitates three- dimensional cell growth, allowing cells to proliferate on the entire surface perimeter as well as within the pores of the scaffold. This design ensures that every cell is in contact with adjacent cells, allowing growth of cells as multilayers, hence promoting cellular interactions and tissue formation as further described herein.

[0189] In accordance with the present disclosure, the cells and liquid (e.g. cell media) may penetrate into the scaffold pores such that the cells may be embedded within the pores of the scaffolds. The scaffold may include pores of varying sizes, distributed throughout its structure.

[0190] These pores are considered to play a critical role in the scaffold's functionality, allowing for the infiltration and growth of cells, as well as the diffusion of nutrients and waste products. The variability in pore sizes may allow a balance between structural integrity and biological compatibility, optimizing the scaffold for its intended use.

[0191] Without being bound by theory, it was suggested that the pores of the scaffold may accommodate attachment and growth of cells within the pores.

[0192] In some embodiments, the porous scaffold comprises a porous material comprises pores wherein the pores have a size that can accommodate at least one cell, at times at least two cells, at times at least ten cells.

[0193] Scaffold pore size can be measured by any method known in the art, for example according to the method described in: (Engineering Aligned Skeletal Muscle Tissue Using Decellularized Plant-Derived Scaffolds I ACS Biomaterials Science & Engineering). Briefly, scaffolds are stained for Calcofluor White M2R (1: 10,000) to stain the cellulose, and imaged at laser scanning confocal microscope to obtain 3D reconstructions of the laden scaffolds. Cross-section XZ profiles of the surface topography of the scaffolds orthogonal to the direction of alignment, are generated from the 3D confocal z-stacks of the Calcofluor White stained cellulose.

[0194] The pore size may be measured by any method known in the field. For example, pore size may be determined by measuring the diameter of a representative sample of pores and determining the mean (average) value.

[0195] The pore size may be measured by any method known in the field. For example, pore size may be determined by measuring the diameter of a representative sample of pores and determining the mean (average) value.

[0196] When referring to a pore size it should be understood as referring to an average pore size of a scaffold structure.

[0197] In some examples, the porous scaffold comprises a porous material comprises pores having a size (diameter) of at least about 2pm, at least about 10pm, at least about 15pm. In some examples, the porous scaffold comprises a porous material comprises pores having a size (diameter) of at least about 20pm, at times at least about 30pm, at times at least about 40pm, at times at least about 50pm, at times at least about 60pm, at times at least about 70pm, at times at least about 80pm, at times at least about 90pm, at times at least about 100pm, at times at least about 110pm, at times at least about 120pm, at times at least about 130pm, at times at least about 140pm, at times at least about 150pm, at times at least about 160pm, at times at least about 170pm, at times at least about 180pm, at times at least about 190pm, at times at least about 200pm, at times at least about 210pm, at times at least about 220pm, at times at least about 230pm, at times at least about 240pm, at times at least about 250pm, at times at least about 260pm, at times at least about 270pm, at times at least about 280pm, at times at least about 290pm, at times at least about 300pm, at times at least about 350pm, at times at least about 370pm, at times at least about 400pm.

[0198] In some examples, the porous scaffold comprises a porous material comprises pores having a diameter between about 2pm and about 2 mm. In some examples, the porous scaffold comprises a porous material comprises pores having a diameter between about 10pm and about 1mm, at times between about 20pm and about 800pm, at times between about 20pm and about 700pm, at times between about 20pm and about 600pm, at times between about 20pm and about 500pm, at times between about 20pm and about 400pm, at times between about 20pm and about 400pm, at times between about 20pm and about 300pm, at times between about 20pm and about 300pm.

[0199] In some examples, the porous scaffold comprises a porous material comprises pores having a diameter of about 2pm, at times about 10pm, at times about 15pm. In some examples, the porous scaffold comprises a porous material comprises pores having a diameter of about 20pm, at times about 30pm, at times about 40pm, at times about 50pm, at times about 60pm, at times about 70pm, at times about 80pm, at times about 90pm, at times about 100pm, at times about 110pm, at times about 120pm, at times about 130pm, at times about 140pm, at times about 150pm, at times about 160pm, at times about 170pm, at times about 180pm, at times about 190pm, at times about 200pm, at times about 210pm, at times about 220pm, at times about 230pm, at times about 240pm, at times about 250pm, at times about 260pm, at times about 270pm, at times about 280pm, at times about 290pm, at times about 300pm, at times about 350pm, at times about 370pm, at times about 400pm.

[0200] As appreciated, the porosity of the scaffolds (i.e. the number of pores / voids in the scaffold structure) is important and it can influence flow of nutrients, gas and waste products, and hence affect the scaffold functionality.

[0201] Porosity can be measured and calculated by different methods known in art, including water retention capacity methods, dry porosity measurement methods and combinations of same.

[0202] For example, scaffold porosity can be determined using the following Formula:

[0203] Scaffold Wet weight- scaffold Dry weight

[0204] Scaffold pore volume _ Water density _

[0205] Scaffold’s porosity =

[0206] Scaffold total volume Scaffold total volume

[0207] In some examples, the scaffold porosity is at least about 40%, at times at least about 50%, at times at least about 55%, at times at least about 60%, at times at least about 65%, at times at least about 70%.

[0208] In some examples, the scaffold porosity is between 50% and 99%, at times between 50% and 95%, at times between 50% and 90%, at times between 50% and 85%, at times between 50% and 80%, at times between 50% and 75%, at times between 50% and 70%.

[0209] In some examples, the scaffold porosity is about 50%, at times about 55%, at times about 60%, at times about 63%, at times about 65%, at times about 70%. In some examples, the scaffold porosity is about 63%.

[0210] In addition, the scaffold water retention capacity can be determined by the bulk density method or the water saturation method. In some examples, water retention capacity is calculated as: water retention capacity = (scaffold wet weight- scaffold dry weight)*100 / (scaffold wet weight).

[0211] In some examples, the plurality of porous three-dimensional scaffold structures have a water retention capacity ranging from between about 50% and about 99% void volume out of a total volume of said three-dimensional structure total volume, calculated as: (scaffold wet weight- scaffold dry weight)* 100 / (scaffold wet weight).

[0212] In some examples, the scaffold water retention capacity is between about 55% and about 99%, at times between about 60% and about 99%, at times between about 65% and about 99%, at times between about 70% and about 99%, at times between about 75% and about 99%, at times between about 80% and about 99%, at times between about 80% and about 98%.

[0213] In some examples, the water retention capacity is about 50%, at times about 55%, at times about 60%, at times about 65%, at times about 70%, at times about 75%, at times about 80%, at times about 85%, at times about 90%, at times about 95%, at times about 96% at times about 97%.

[0214] In some examples, the scaffold water retention capacity is about 75%, at times about 80%, at times about 85%, at times about 90%, at times about 95%, at times about 96% at times about 97%.

[0215] Packing dry density as used provides a measure of how tightly the scaffolds are packed together in dry configuration and can be defined as the mass of dry scaffolds divided by the scaffolds volume under conditions at which the scaffolds are tightly packed or compacted (and without addition of water or any other fluid).

[0216] In some examples, the plurality of porous three-dimensional scaffold structures are packed with a packing dry density ranging from between about Img / ml and about lOOOmg / ml, at times between about Img / ml and about 750mg / ml, at times between about Img / ml and about 500mg / ml, at times between about Img / ml and about 250mg / ml, at times between about Img / ml and about lOOmg / ml, at times between about Img / ml and about 75mg / ml, at times between about Img / ml and about 50mg / ml, at times between about Img / ml and about 40mg / ml, at times between about 2mg / ml and about 40mg / ml, at times between about 3mg / ml and about 30mg / ml, at times between about 4mg / ml and about 25mg / ml, at times between about 5 mg / ml and about 20mg / ml. .

[0217] Packing wet density as used herein provides a measure of how the scaffolds are packed together when they are wet or immersed in a liquid. In some examples, the plurality of porous three-dimensional scaffold structures are packed with a packing wet density ranging from between about 0.1 g / ml and about 1.0 g / ml, at times between about 0.1 g / ml and about 0.75g / ml, at times between about 0.1 g / ml and about 0. 5g / ml.

[0218] As noted herein, the scaffold is not limited to a specific shape and may be in any shape that allows the packing of the plurality of the scaffolds in the bioreactor. In some examples, the three-dimensional scaffold structures have a shape selected from the group consisting of sheet, fiber, bead, flak, disc, sphere, cylinder, ring, polygon and star shape.

[0219] In some examples, the three-dimensional scaffold structures have a square shape. In some examples, the three-dimensional scaffold structures have a circle shape. In some examples, the three-dimensional scaffold structures have a hexagonal shape. In some examples, the three-dimensional scaffold structure does not have a fiber structure. In some examples, the three-dimensional scaffold is not a hollow fiber scaffold. In some examples, the bioreactor is not a hollow fiber bioreactor.

[0220] The scaffold in accordance with the present disclosure is configures to allow cell density of 20- 100k cells / mg wet scaffold.

[0221] In accordance with the present disclosure, the edible scaffold is characterized by their ability to provide texture and color to the formed alterative food.

[0222] As shown in Figure 13, cultivated meat product that was prepared in the EBP (with 6.8% cells) is characterized by a higher organoleptic effect as compared to a hybrid product composed of the same % of cell slurry. In addition, cultivated meat product that was prepared in the EBP (with 6.8% cells) is characterized by the same organoleptic effect of hybrid product composed of 50% cell slurry. These results demonstrate that cell and tissue growth in the EBP is advantageous, as it provides desirable organoleptic properties with a low cell density. This suggests that the increased productivity of cells and tissue production in the EBP is associated with the release of volatile compounds that mimic the sensory characteristics of traditional food products.

[0223] As described herein, the plurality of scaffolds within the packed bed bioreactor are suitable to support cell attachment, cell growth and proliferation and / or differentiation. In some examples, the packed bed bioreactor is for use with a source of culture media for growing cells within the basin.

[0224] In order to increase the effectiveness of the scaffolds, the scaffolds may be pretreated scaffold, i.e. treated prior to cell seeding.

[0225] These treatments may involve modifying the scaffold's surface and / or pores property, including, inter alia, mechanical strength and surface characteristics, in order to enhance their performance and suitability for cells growth and / or cell differentiation.

[0226] In some examples, the porous three-dimensional scaffold structures are preconditioned. In some examples, the porous three-dimensional scaffold structures are preconditioned to promote adherence of cells onto said scaffold structures.

[0227] Treatment of the scaffolds prior to cell seeding is denoted herein as preconditioning.

[0228] In some examples, the pre-conditioning comprises physical and / or chemical change of surface of the porous three-dimensional scaffold structures.

[0229] In some examples, the pre-conditioning comprises physical change of surface of the porous three-dimensional scaffold structures. As used herein the term physical change of the surface of porous three-dimensional scaffold structures refers to any alteration or modification that occurs in the physical properties of at least one surface of the scaffolds while maintaining their porous and three-dimensional nature. A physical property may include one or more of texture or topography of the scaffold surface and a change in one or more of the physical properties may be achieved, for example by surface roughening, smoothing, patterning, or etching.

[0230] In some examples, the pre-conditioning comprises chemical change of surface of the porous three-dimensional scaffold structures. As used herein the term chemical change of surface of the porous three-dimensional scaffold structures refers to any alteration or modification that occurs in the surface chemistry and composition of the porous three- dimensional scaffold structure, for example by introducing new chemical functionalities or changing existing functionalities of the scaffolds while maintaining their porous and three- dimensional nature. The chemical changes may involve attaching chemical substances, molecules, or polymers to the scaffold surface via covalent bonding or non-covalent bonding.

[0231] In some examples, the pre-conditioning comprises any one of plasma treatment, electrostatic charging of surface of the porous three-dimensional scaffold structures; increasing surface energy of the porous three-dimensional scaffold structures, removing lignin from surface of the porous three-dimensional scaffold structures; exposing cellulose residues on surface of the porous three-dimensional scaffold structures.

[0232] In some examples, the pre-conditioning comprises plasma treatment.

[0233] In some examples, the porous three-dimensional scaffold structures are preconditioned to expose cellulose residues on the surface of the porous three-dimensional scaffold structures.

[0234] In some examples, the porous three-dimensional scaffold structures are preconditioned to remove lignin from surface of the porous three-dimensional scaffold structures.

[0235] In some examples, the porous three-dimensional scaffold structures are preconditioned using vacuum plasma treatment or corona treatment.

[0236] In some examples, the porous three-dimensional scaffold structures are preconditioned using vacuum plasma treatment.

[0237] In some examples, the porous three-dimensional scaffold structures are plasma treated scaffolds.

[0238] As shown in Figure 4, cell growth and protein content were increased in the plasma treated scaffolds.

[0239] In some examples, the porous three-dimensional scaffold structures are preconditioned to increase the positive charge of the surface of the porous three-dimensional scaffold structures.

[0240] Regardless of the pre-conditioning that is used, it should be noted that the preconditioning is determined by level of adherence of cells onto the porous three-dimensional scaffold structures. The pre-conditioning may be determined by assessing the positive charge of the surface and the % exposed cellulose binding sites. Cellulose binding sites can be measured by any method know in the art, for example by fluorescence-based assays in which fluorescently labeled cellulose-binding molecules may be used to measure cellulose binding sites.

[0241] In some examples, the porous three-dimensional scaffold structures are washed with an attachment solution.

[0242] In some examples, the porous three-dimensional scaffold structures undergo washing subsequent to the pre-conditioning.

[0243] In some examples, the porous three-dimensional scaffold structures are washed with an attachment solution.

[0244] The attachment solution as used herein refers to a cell culture medium that enhances / facilitates attachment / adhesion of cells to the porous scaffold surface. As described herein, the attachment solution is used to maximize adherence of the cells to the scaffolds surface.

[0245] The attachment solution comprises one or more biomolecules or derivatives thereof that are capable of interacting with cell surface receptors and hence may be considered as attachment factors to enhance interactions with cells.

[0246] In some examples, the attachment solution is selected from the group consisting of fibronectin attachment solution, gelatin attachment solution, collagen attachment solution, laminin attachment solution, vitronectin attachment solution, elastin attachment solution poly-L-lysine attachment solution, gelatin attachment solution or spent media attachment solution, derivatives or combinations thereof.

[0247] In some examples, there is no attachment material, and the scaffolds are washed with PBS or fresh media.

[0248] It should be noted that by using one or more of the attachment solutions facilitates the attachment and spreading of cells on the surface of the scaffolds.

[0249] As shown in Figure 5, washing the scaffold with attachment solution or spent media, prior to cell seeding effectively enhanced the scaffold's ability to support cell attachment and proliferation, in some cases, washing the scaffolds with PBS is sufficient for cell attachment and proliferation.

[0250] In some examples, the porous three-dimensional scaffold structures are lyophilized structures.

[0251] In some examples, the porous three-dimensional scaffold structures are sterilized. The scaffolds can be sterilized at any time, for example before being packed within the bioreactor, or after being introduced within the bioreactor.

[0252] In some examples, the porous three-dimensional scaffold structures are sterilized within the bioreactor.

[0253] Sterilization can be done by any method known in the art. In some examples, the porous three-dimensional scaffold structures are sterilized by any sterilization method selected from heat by steam or autoclaving, UV irradiation, radiation sterilization and dry heat sterilization.

[0254] In some examples, the bioreactor with the scaffolds within are sterilized using dry autoclave.

[0255] As detailed above, the plurality of scaffolds are placed within the bioreactor to allow any one of cell attachment, growth, differentiation and tissue formation.

[0256] As described herein, the bioreactor's applicability is highly versatile, as it can accommodate a single scaffold type as well as a variety of scaffold types in different distributions, each tailored to support different purposes and enhance tissue production. This flexibility allows for the integration of multiple scaffold types within the same bioreactor, which can be arranged to create a homogenous or a heterogeneous (non- homogenous, organized) environment that optimally supports the growth of various tissues.

[0257] For example, as described hereinbelow, scaffolds designed for muscle tissue growth can be arranged to provide the necessary rigidity and structure, while scaffolds intended for fat tissue formation can be positioned to ensure flexibility and nutrient availability. Additionally, as these different scaffold types can be distributed in either a homogeneous or non-homogeneous (organized) manner, different cultivated food product can be produced.

[0258] This capability enables the bioreactor to produce a variety of food products including multi-textured food products.

[0259] The scaffolds, either the same or different, may be assembled in several configurations within the bioreactor. Reference is made to Figure 1A that is a schematic illustration showing a bioreactor with homogeneous distribution of a single type of scaffold structures. Figure IB and Figure 1C show, respectively, bioreactor with two different types of scaffolds structures with different distribution within the bioreactor, with Figure IB showing essentially homogenous distribution / mixture of two different scaffolds in the bioreactor, while Figure 1C shows zones within the bioreactor such that each scaffold structure occupies a different zone (unmixed population of scaffold structures).

[0260] Hence, the plurality of scaffold structures may comprise a single type of scaffold structures or at least two different types of scaffolds structures.

[0261] In some examples, the plurality of scaffold structures comprises a single type of scaffold. In some examples, the plurality of scaffold structures comprises a single type of scaffold is arranged within the bioreactor in a homogenous distribution. A homogeneous distribution of a single type of scaffold is shown in Figure 1A. In some examples, the plurality of scaffold structures comprises a single type of scaffold may be used for producing a single type of cells. The selection of the single type of scaffold depends on the required type of cells and tissue.

[0262] In some examples, the bioreactor comprises a plurality of scaffold structures, characterized by an elastic modulus of at most about lOOkPa, at times between about 3kPa and about 100 kPa. In some examples, the plurality of scaffold structures, characterized by an elastic modulus of at most about lOOkPa, at times between about 3kPa and about 100 kPa is suitable for growth of adipose, liver or connective tissues.

[0263] In some examples, the bioreactor comprises a plurality of scaffold structures, characterized by an elastic modulus of at least about lOOkPa, at times between about lOOkPa and about 600 kPa. In some examples, the plurality of scaffold structures, characterized by an elastic modulus of at least about lOOkPa, at times between about lOOkPa and about 600 kPa is suitable for growth of muscle, cartilage or bone tissues.

[0264] It should be noted that the use of different types of scaffolds may be useful in supporting the growth and / or differentiation of different cell types. Hence, a diverse range of scaffolds with different properties, such as pore size, porosity, surface chemistry, and mechanical strength, may be used to provide an optimal environment for growth and / or differentiation of different cell types within the packed bed bioreactor.

[0265] When referring to different types of scaffolds it should be understood such that the scaffold defined as having different types are different in at least one scaffold property, including, inter alia, scaffold material, scaffold size, scaffold shape, scaffold stiffness, scaffold elasticity, scaffold average pore size, scaffold porosity, scaffold color or scaffold texture.

[0266] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types differ in at least one, at times at least two, at times at least three of scaffold material, scaffold size, scaffold shape, scaffold stiffness, scaffold elasticity, scaffold average pore size, scaffold porosity, scaffold color, scaffold texture or any combination thereof.

[0267] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types have at least different edible material.

[0268] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types have at least different scaffold size.

[0269] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types have at least different scaffold shape. In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types have at least different scaffold stiffness.

[0270] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types have at least different scaffold elasticity.

[0271] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types have at least different scaffold average pore size.

[0272] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types have at least different scaffold porosity.

[0273] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types have at least different scaffold color.

[0274] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein the at least two different types have at least different scaffold texture.

[0275] The arrangement of the plurality of scaffolds within a bioreactor may vary depending on multiple considerations, including, inter alia, tissue to be formed, bioreactor configuration, cultivated product texture, cultivated product color.

[0276] In some examples, the plurality of scaffold structures is arranged in a homogenous distribution.

[0277] In some examples, the plurality of scaffold structures comprises a single type of scaffold, wherein said scaffold structures are arranged in a homogenous distribution.

[0278] In some examples in which the plurality of scaffold structures comprises at least two different types of scaffolds, these scaffolds may be arranged in a homogenous mixture distribution within the bioreactor. As used herein, a homogenous mixture distribution (arrangement) of a single type of scaffold or at least two types of different scaffold in the bioreactor refers to an even and consistent arrangement of that specific scaffold throughout a given space or medium within a bioreactor. This means that the scaffold is uniformly spread, ensuring that its properties and functions are equally accessible in all areas of the bioreactor. In case of two or more different types of scaffolds, the different scaffold types are uniformly dispersed, ensuring that each type is evenly distributed and occupies the same proportion of space throughout the entire volume or area.

[0279] A homogeneous mixture of two different types of scaffolds is shown in Figure IB.

[0280] In some examples, the plurality of scaffold structures comprises at least two different types of scaffolds, wherein said scaffolds are arranged non-homogeneously within the bioreactor.

[0281] As used herein an organized distribution (arrangement) of two or more types of different scaffolds refers to a to an intentional tailored made arrangement where the various scaffold types are not evenly dispersed throughout the bioreactor. In other words, the various different scaffold types are placed in specific regions to optimize the bioreactor's functionality and meet specific biological and mechanical requirements.

[0282] An organized distribution of two different types of scaffolds is shown in Figure 1C.

[0283] In some examples, the arrangement of the at least two different types of scaffolds depend on the produced tissue and hence alternative food product.

[0284] In some examples, the bioreactor comprises a plurality of scaffold structures, including at least two different types of scaffolds with distinct elasticities, arranged in a homogeneous distribution.

[0285] In some examples, the bioreactor comprises a plurality of scaffold structures, including at least two different types of scaffolds, wherein at least one type of scaffold is characterized by an elastic modulus of at most about lOOkPa and at least one other different type of scaffold is characterized by an elastic modulus of at least about lOOkPa.

[0286] In some examples, the bioreactor comprises a plurality of scaffold structures, including at least two different types of scaffolds, wherein at least one type of scaffold is characterized by an elastic modulus of between about 3kPa and about 100 kPa and at least one other different type of scaffold is characterized by an elastic modulus of between about lOOkPa and about 600 kPa.

[0287] In some examples, the scaffold is characterized by an elastic modulus of at most about lOOkPa may be suitable for growth and differentiation of cells into soft tissue.

[0288] In some examples, the scaffold is characterized by an elastic modulus of at most about lOOkPa may be suitable for growth and differentiation of adipose, liver or connective tissues. In some examples, the scaffold is characterized by an elastic modulus of between about 3kPa and about lOOkPa may be suitable for growth and differentiation of adipose, liver or connective tissues.

[0289] In some examples, the scaffold is characterized by an elastic modulus of at least about lOOkPa may be suitable for growth and differentiation of cells into rigid tissue.

[0290] In some examples, the scaffold is characterized by an elastic modulus of at least about lOOkPa may be suitable for growth and differentiation of muscle or bone cells. In some examples, the scaffold is characterized by an elastic modulus of between about lOOkPa and about 600kPa may be suitable for growth and differentiation of muscle and bone cells.

[0291] In case of a homogeneous distribution of these scaffolds with different elasticity both strong and flexible tissue components can be developed uniformly throughout the bioreactor. Such a combination of scaffolds supporting both muscle, fat and bone tissue growth, may provide structure and alignment for muscle fibers together with adipose and bone cells.

[0292] In some examples, the bioreactor comprises a plurality of scaffold structures, including at least two different types of scaffolds having different elasticity may be arranged in a non-homogenous organized distribution in the bioreactor. In some examples, the scaffold is characterized by an elastic modulus of at most about lOOkPa may accommodate inner parts of the bioreactor and the scaffold is characterized by an elastic modulus of at least about lOOkPa may accommodate top and bottom parts of the bioreactor. In some examples, the bioreactor comprises a plurality of scaffold structures, including at least two different types of scaffolds having different sizes may be arranged in homogeneous and / or non-homogenous organized distribution in the bioreactor. In some examples, the scaffold is characterized by a scaffold size of at most 6mm may accommodate inner parts of the bioreactor and the scaffold is characterized by a scaffold size of at least 6mm may accommodate top and bottom parts of the bioreactor to support the mechanical load and functioning of the packed bed, and to enable media flow in the void volume.

[0293] In some examples, the bioreactor comprises a plurality of scaffold structures, including at least two different types of scaffolds having different color. In some examples, the bioreactor comprises a plurality of scaffold structures, including at least two different types of scaffolds having different color are determined such that each one of the scaffold types may resemble the produced tissue for food alternative.

[0294] The packing characteristics, such as packing density, of the porous scaffold structures in the packed bed bioreactor may depend on the porosity of the scaffolds and may affect the activity and performance of the bioreactor in growing and differentiating cells. The packing density that is typically defined as the volume of scaffold structures per total volume of the bioreactor, can be measured under dry conditions or under wet conditions.

[0295] The bioreactor described as shown for example in Figure 12A has a first end (120) and a second end (140) and an internal space (160) filled with plurality of porous three- dimensional scaffold structures (180).

[0296] In some examples, the at least one fluid inlet and / or the at least one fluid outlet form an integral part of the bioreactor.

[0297] The fluid inlet is configured to deliver media with or without cells into the bioreactor and the fluid outlet is configured to remove waste media away from the bioreactor. The packed bed reactor may include additional components. In some examples, the packed bed bioreactor comprises a removable headplate configured to allow introduction and / or removal of content of the basin.

[0298] As described herein, the packed bed bioreactor comprises means to determine the condition of the cells throughout the growth and / or differentiation. Hence, the bioreactor comprises means allowing monitoring the condition, for example, by specific sensors. In some examples, the packed bed bioreactor comprises at least one port for accommodating at least one sensor.

[0299] The present disclosure is not limited to a specific sensor and the bioreactor can accommodate one or more sensors that is configured for monitoring the status of the cells by determining at least one media parameter.

[0300] In some examples, the at least one sensor is selected to detect at least one parameter selected from the group consisting of pH, dissolved oxygen (DO), temperature, conductivity, capacitance, glucose, lactate, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH), biomass weight, biomass level in basin, foam.

[0301] In some examples, one or more sensors is a sterilized sensor. In some examples, the one or more sensors can be sterilized within the bioreactor. In some examples, the one or more sensors can be sterilized and afterwards placed within the bioreactor.

[0302] In some examples, the sensors are sterilized separately and then placed aseptically in the bioreactor. In some examples, the pH and dissolved oxygen (DO) sensors are sterilized separately, and then placed aseptically in the bioreactor. In some examples, the pH and dissolved oxygen (DO) sensors are autoclaved.

[0303] The bioreactor comprises, in accordance with some examples, a mechanism configured to allow fluid circulation within the internal space of the bioreactor. In some examples, the mechanism comprises at least one component selected from the group consisting of impeller, peristaltic pump, diaphragm pump, vibrator, tilter and lifter (hydraulic head).

[0304] The fluid circulation may depend on the size of the bioreactor and the number of scaffolds and cells deposited within the bioreactor. In some examples, the fluid circulation is range between about 0.1-3 bioreactor liquid medium working volume replacements in a minute.

[0305] The bioreactor is configured to allow flow of fluids via the inlet into the basin. In some examples, the basin is positioned in an orientation to allow flow up of fluids from the fluid inlet upwards, and through the packed bed.

[0306] As used herein the term flow encompasses medium flow refers to a configuration of the bioreactor in which the medium that enters through the inlet is in close contact with the scaffolds and cells within the bioreactor.

[0307] The bioreactor comprises at least one control loop. The at least one control loop is configured to maintain at least one parameter selected from the group consisting of pH, dissolved oxygen and temperature at a predetermined setpoint value. The setpoint value is determined to allow growth and / or differentiation of cells.

[0308] In some examples, the basin is made of a biocompatible material. In some examples, the basin is made of a polymer. In some examples, the polymer is one or more of Polypropylene, Polyvinyl Chloride, Polycarbonate, Polystyrene.

[0309] In some examples, the basin is made of glass. In some examples, the basin is made of stainless steel.

[0310] In some examples, the basin comprises at least one transparent segment in the wall, configured to allow viewing content of the internal space.

[0311] The scaffolds are packed within the packed bed bioreactor and serve as a platform of cell seeding, cell growth and / or cell differentiation as well as tissue formation.

[0312] The present disclosure is not limited to specific cell types or a specific source of cells and is applicable to various cell types, provided that they have the ability to attach to the plurality of scaffolds.

[0313] As shown in Figures 6A-6H, several cell types were able to adhere to and proliferate on the scaffold.

[0314] In some examples, the cells are adherent cells. In some examples, the cells are non-human cells. In some examples, the cells are from an animal (non-human) source. In some examples, the cells are adherent animal (non- human) cells. In some examples, the cells are non-genetically modified. In some examples, the cells are genetically modified.

[0315] As described herein, in accordance with some examples, the cells are used for the production of alternative cultured food product, such as alternative cultured meat or alternative cultured fish and sea food.

[0316] In some examples, the cells are from a mammalian.

[0317] In some examples, the cells are from a non-human mammalian. In some examples, the cells are non-human cells.

[0318] In some examples, the cells are from poultry.

[0319] In some examples, the cells are from avian. In some examples, the cells are from avian eggs. In some examples, the cells are avian cells. In some examples, the cells are avian egg cells.

[0320] In some examples, the cells are any one of chicken cells, duck cells or turkey cells.

[0321] In some examples, the cells are from a bovine.

[0322] In some examples, the cells are from a cow, a pig, a sheep, a rabbit, an equine, a canine, or any combination thereof.

[0323] In some examples, the cells are obtained from a placenta. In some examples, the cells are placenta cells.

[0324] In some examples, the cells are obtained from am embryo.

[0325] In some examples, the cells are chicken embryonic fibroblast cells.

[0326] In some examples, the cells are duck embryonic fibroblast cells. In some examples, the cells are from an aquatic animal.

[0327] In some examples, the cells are from one or more of salmon, tuna, shrimp or lobster.

[0328] In some examples, the cell comprises at least stem cells. Stem cells as used herein refers to undifferentiated cells that have the ability to develop into various specialized cell types.

[0329] In some examples, the cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, pluripotent stem cells, adult stem cells, mesenchymal stem cells, myoblasts, satellite muscle cells, fibroblasts, hepatoblasts, osteoblasts, chondroblasts, adipocytes, hepatocytes, osteocytes, chondrocytes, and any combination of same, and any other natural and genetically modified adherent cell.

[0330] In some examples, the cells are embryonic poultry stem cells. In some examples, the cells are embryonic duck stem cells. In some examples, the cells are embryonic bovine stem cells.

[0331] In some examples, the cells are poultry Adipose-derived Stem (pASC) cells. In some examples, the cells are poultry fibroblast cells.

[0332] In some examples, the cells are bovine ASC cells (bASCs). In some examples, the cells are bovine fibroblast cells.

[0333] As described herein, for the purpose of cells growth and / or differentiation, cells are allowed to grow on the scaffold structures.

[0334] In some examples, the cells may be seeded on the scaffolds within the bioreactor. In other words, the scaffolds are first placed in the bioreactor and then cell are added to the bioreactor to allow contact between the cells and scaffolds and seeding of the cells onto the scaffold and within the scaffold pores as described herein.

[0335] Hence, in accordance with a second aspect of the presently disclosed subject matter there is provided a method comprising:

[0336] (i) introducing cell media comprising cells into a packed bed bioreactor, the bioreactor comprising a basin having a first end, a second end and a wall extending between the first end and the second end defining an internal space, the bioreactor further including at least one fluid inlet at the first end and at least one fluid outlet; wherein the internal space is filled with plurality of porous three- dimensional scaffold structures; wherein the plurality of porous three-dimensional scaffold structures comprises edible material; wherein the three-dimensional scaffold structures are packed within the basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of the porous three-dimensional scaffold structures within the internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet;

[0337] (ii) providing conditions that support growth and / or differentiation of the cells on the porous three-dimensional scaffold structures; and

[0338] (iii) harvesting the porous three-dimensional scaffold structures with cells thereon.

[0339] As described herein, in order to increase cell attachment and hence maximize the seeding of the cells onto and within pores of the scaffold, the scaffold may be treated prior to cells seeding. In such cases, the method may comprise a step of treating the scaffold prior to cell seeding. Treatment of the scaffolds prior to cell seeding is denoted herein as pre-conditioning.

[0340] In some examples, the method comprising pre-conditioning of the scaffold. In some examples, the pre-conditioning comprises plasma treatment. In some examples, the preconditioning comprises vacuum plasma treatment.

[0341] In some examples, the method comprises subjecting the plurality of scaffold structures to pre-conditioning prior to cell seeding.

[0342] In some examples, the method comprises subjecting the plurality of scaffold structures to at least one pre-conditioning step prior to cell seeding, wherein the preconditioning is done outside of the bioreactor. In other words, the plurality of scaffold structures are subjected to at least one pre-conditioning step prior to being introduced and packed within the bioreactor.

[0343] In some examples, the method comprises subjecting the plurality of scaffold structures to at least one pre-conditioning step prior to cell seeding, wherein the preconditioning is within the bioreactor. In other words, the plurality of scaffold structures are introduced and packed within the bioreactor and thereafter subjected to at least one preconditioning step.

[0344] In some examples, the method comprises subjecting the plurality of scaffold structures to plasma treatment prior to cell seeding.

[0345] In some examples, the method comprises subjecting the plurality of scaffold structures to plasma treatment prior to cell seeding, wherein the plasma treatment is conducted outside of the bioreactor. In other words, the plurality of scaffold structures are subjected to plasma treatment prior to being introduced and packed within the bioreactor.

[0346] In some examples, the method comprises subjecting the plurality of scaffold structures to plasma treatment prior to cell seeding, wherein the plasma treatment is conducted within the bioreactor. In other words, the plurality of scaffold structures are introduced and packed within the bioreactor and thereafter subjected to plasma treatment.

[0347] In some examples, the method comprises subjecting the plurality of scaffold structures to at least one washing step prior to cell seeding.

[0348] In some examples, the method comprises subjecting the plurality of scaffold structures to at least one washing steps subsequent to pre-conditioning.

[0349] In some examples, the method comprises subjecting the plurality of scaffold structures to at least one pre-conditioning step and to at least one washing steps.

[0350] In some examples, the method comprises subjecting the plurality of scaffold structures to at least one washing steps subsequent to plasma treatment.

[0351] In some examples, the method comprises subjecting the plurality of scaffold structures to plasma treatment and to at least one washing steps.

[0352] The at least one washing step may be performed with any of the attachment solutions described herein.

[0353] In some examples, the method comprises sterilizing the scaffolds. Sterilization may be performed by any method described herein.

[0354] As noted herein, the method comprises introducing of cell media comprising cells into the packed bed bioreactor. The cell media as used herein also known as cell culture media, refers to a nutrientrich solution that in accordance with the present disclosure is used to support one or more of growth, proliferation, and differentiation of cells.

[0355] The methods of the present disclosure comprise introducing cell media into the bioreactor such that the cell media comprises cells.

[0356] As described herein, the present disclosure is applicable for a variety of cells and hence the specific composition of the cell media may be adjusted to support such cells. Hence, the suitable cell media may depend on the type of cells and typically include growth factors, antibiotics, nutrients or any combination thereof.

[0357] In some examples, the cell media comprises a single cell type.

[0358] In some examples, the cell media comprises more than one cell type.

[0359] In some examples, the cell media comprises two or more types of non-human cells. It should be noted that the selection of the one or more cell type that are used may depend on the differentiation potential of the cells and the alternative food to be produced, for example meat as well as meat portion to be produced.

[0360] It should be noted that in some cases where more than one cell type is used, different types of scaffolds may be placed in the bioreactor to support the growth and function of these different cells.

[0361] In some examples, the cell media comprises at least stem cells.

[0362] In some examples, the cell media comprises at least cells selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, pluripotent stem cells, adult stem cells, mesenchymal stem cells, myoblasts, satellite muscle cells, fibroblasts, hepatoblasts, osteoblasts, chondroblasts, adipocytes, hepatocytes, osteocytes, chondrocytes, and any combination of same, and any other natural and genetically modified adherent cell.

[0363] In some examples, the cell media comprises one or more of poultry ASC cells, poultry fibroblast cells, bovine ASC cells, bovine fibroblast cells or any combination thereof. In some examples, the cell media comprises one or more of stromal cells, endothelial cells, fat cells, hematopoietic cells, myocytes, hepatocytes, chondrocytes, osteocytes and fibroblast.

[0364] The number of cells in the cell media depends on multiple factors, including, inter alia, the number of scaffolds, their spatial orientation, and their properties (e.g. porosity). As appreciated, the higher the porosity of the scaffold it would be possible to increase the number of cells to be seeded on the scaffolds and accordingly the number of cells in the cell media being introduced into the bioreactor.

[0365] In accordance with some examples, the method comprises introducing cell media via at least one fluid inlet of the bioreactor.

[0366] In some examples, the method comprises introducing cell media via at least one fluid inlet under conditions that support seeding and growth of cells.

[0367] After the cell media including the cells have been introduced into the bioreactor, the cells are allowed to attach to the scaffold (i.e. seeding of cells) and growth under conditions allowing such growth.

[0368] The conditions that allow or support cell growth on the porous three-dimensional scaffold structure and within the pores encompasses conditions supportive of cell seeding and conditions supporting of cell growth.

[0369] Cell seeding as used herein refers to attachment / adherence of the cells to the surface of the scaffolds and / or into the pores of the surface and at times is referred herein as the seeding phase.

[0370] The number of the cells seeded depends for example on the scaffolds density and is determined such that the cells are at least partially covering the at least one surface of the plurality of scaffolds and / or at least partially covering the at least a part of the voids / pores of the plurality of scaffolds.

[0371] In some examples, the seeding may comprise one or more steps of cell seeding. In some examples, the method comprises a single seeding step. In some examples, the method comprises sequential seeding steps. In some examples in which the method comprises sequential cell seeding steps, the cells in each step may be the same or may be different. In some examples, the conditions supportive of cell seeding may include flow velocity, cell concentration and medium volume.

[0372] In some examples, the method comprises introducing the cell media at a flow velocity between about 1 cm / sec and 5cm / sec. In some examples, the method comprises introducing the cell media at a flow velocity of about 1 cm / sec, at times about 2 cm / sec, at times about 3 cm / sec, at times about 4 cm / sec, at times about 5 cm / sec. As appropriated, such flow velocity allows attachment of cells to the scaffold structure (onto and within the pores) and hence suitable for cell seeding.

[0373] During cell seeding, agitation or vibration may be applied to maximize the homogeneity of the seeding. The rotational speed and the time of agitation or vibration may vary depending on the number of cells and scaffolds introduced into the bioreactor.

[0374] In some examples, the method comprising applying vibration at an amplitude of between about 0.1 cm and about 10 cm. In some examples, the method comprising applying vibration at a frequency between about 0.1 Hz and about 4Hz. In some examples, the method comprising applying vibration continuously. In some examples, the method comprising applying vibration intermittently.

[0375] In some examples, the method comprising applying agitation during the seeding. In some examples, the method comprises agitation at one rotational speed. In some examples, the method comprises agitation at two rotational speeds.

[0376] In some examples, the method comprising agitation at a first rotational speed in a range of between about lOrpm and about 50rpm. In some examples, the method comprising agitation at a first rotational speed of about lOrpm, at times about 20rpm, at times about 30rpm, at times about 40rpm, at times about 50rpm.

[0377] In some examples, the method comprising agitation at a second rotational speed between about lOOrpm and about 200rpm. In some examples, the method comprising agitation at a second rotational speed of about lOOrpm, at times about 130rpm, at times about 150rpm, at times about 170rpm, at times about 200rpm. In some examples, the method comprising agitation at alternating velocities including a first rotational speed in a range of about lOrpm and about 50rpm, and a second rotational speed between about lOOrpm and about 200rpm.

[0378] As noted herein, the time for an agitation step may vary depending on various factors, including, inter alia, the rotational speed.

[0379] In some examples, the method comprising agitation at the first rotational speed is for a period of between about 10 minutes and about 20 minutes. In some examples, the the method comprising agitation at the first rotational speed is for a period of about 10 minutes, at times about 15 minutes, at times about 20 minutes.

[0380] In some examples, the method comprising agitation at the second rotational speed is for a period of less than 1 minute. In some examples, the method comprising agitation at the second rotational speed is for a period of about 10 seconds, at times about 20 seconds, at times about 30 seconds, at times about 40 seconds, at times about 50 seconds.

[0381] In some examples, the method comprising agitation at the first rotational speed is for a period of between 10 minutes and 20 minutes, and the agitation at the second rotational speed is for a period of less than 1 minute.

[0382] As described herein, the cells to be seeded have the ability to growth, i.e. proliferate and differentiate under suitable conditions to generate a variety of cells and tissue.

[0383] The term growth of cells as used herein refers to a stage after the cells have been seeds on at least one surface of the plurality of scaffolds and / or within the pores of the plurality of scaffolds.

[0384] During the growth phase, there is an increase in the number and / or size of cells due to cell proliferation.

[0385] The time period at which the cells are allowed to proliferate in the bioreactor is denoted herein as growth phase.

[0386] In accordance with the present disclosure, the methods provide conditions that support growth of the cells. As appreciated, the conditions during the growth phase are optimized to suit the cells being grown in the bioreactor and may vary depending for example, on the cell type introduced into the bioreactor and include, inter alia, nutrients, temperature, pH, osmolarity, gas exchange. In some examples, the conditions that support growth of the cells on the porous three-dimensional scaffold structures comprise at least one condition selected from the group consisting of temperature, pH, dissolved oxygen (DO) and agitation.

[0387] In some examples, the cell media used in the growth phase is the same media as in the seeding phase. In some examples, the cell media used in the growth phase is different than the media in the seeding phase.

[0388] In some examples, the cell media in the growth phase is suitable for growth of nonhuman animal cells.

[0389] In some examples, the cell media in the growth phase comprises one or more of serum (e.g. fetal bovine serum (FBS)), nutrients, amino acids, antibiotics or any combination thereof.

[0390] In some examples, the cell media in the growth phase comprises DMEM / F-12, FBS, glutamax and Gentamycin.

[0391] In some examples, the method comprises monitoring during cell growth at least one parameter selected from the group consisting of pH, dissolved oxygen (DO), temperature capacitance, metabolites, biomass weight, biomass level in basin and foam.

[0392] In some examples, the metabolite comprises at least one metabolite selected from the group consisting of glucose, lactate, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH).

[0393] In some examples, the metabolite comprises at least one metabolite selected from the group consisting of glucose, lactate, NH3 and lactate dehydrogenase (LDH).

[0394] In some examples, the method comprises monitoring glucose consumption rate.

[0395] In some examples, the method comprises monitoring lactate formation rate.

[0396] In some examples, the method comprises monitoring ammonia formation rate.

[0397] In some examples, the method comprises monitoring LDH formation rate. In some examples, the method comprises applying a temperature within a range of between about 20°C and about 45°C. In some examples, method comprise applying a temperature within a range of between about 25 °C and about 40°C, at times between about 30°C and about 40°C. In some examples, method comprise applying a temperature is about 25°C, at times about 30°C, at times about 35°C, at times about 37°C, at times about 39°C.

[0398] In some examples, the method comprises maintaining a pH within a range of between about 6 and about 8.

[0399] In some examples, the method comprises maintaining a level of DO within a range of between about 10% and about 100%, at times between about 20% and about 90%, at times between about 20% and about 80%, at times between about 30% and about 70%.

[0400] In some examples, the method comprises agitation or vibration with a shear stress of between about O.ldyn / cm2and about 10dyn / cm2, at times between about O.ldyn / cm2and about 5dyn / cm2, at times between about O.ldyn / cm2and about ldyn / cm2.

[0401] In some examples, the method comprises during cell growth vibration at an amplitude of between about 0.1 cm and about 10 cm. In some examples, the method comprises vibration at a frequency between about 0.1 Hz and about 4Hz. In some examples, the vibration is applied continuously. In some examples, the method comprises applying vibration intermittently.

[0402] In some examples, the method comprises agitation during the cell growth at a rotational speed within a range of about lOOrpm and about 200rpm. In some examples, the method comprises agitation at a rotational speed of about lOOrpm, at times about 150rpm, at times about 200rpm.

[0403] In some examples, the method comprises applying flow velocity during cell growth between about 5cm / sec and 30cm / sec. In some examples, the method comprises applying flow velocity during cell growth is about 5cm / sec, at times about lOcm / sec, at times about 15cm / sec, at times about 20cm / sec, at times about 25cm / sec, at times about 30cm / sec.

[0404] As shown in the examples below, an increase in glucose consumption rate along with an increase in lactate, ammonia and LDH formation rate was observed during the growth phase. In addition, an increase was observed in the protein content during this phase.

[0405] As appreciated, when referring to growth of cells it is to be understood as allowing the cells to proliferate such that the % of cell coverage increases.

[0406] The coverage of the scaffold with cells can be expressed as the cell density per scaffold.

[0407] In some examples, the cell density per scaffold is at least about O.lmillion cells per scaffold, at times at least about 0.3million cells per scaffold, at times at least about 0.5million cells per scaffold, at times at least about 0.6million cells per scaffold, at times at least about 0.7million cells per scaffold , at times at least about 0.8million cells per scaffold, at times at least about 0.9million cells per scaffold, at times at least about 1 million cells per scaffold.

[0408] In some examples, the cell density per scaffold is between about 0.1 million cells per scaffold and about 2 million cells per scaffold, at times between about 0.3 million cells per scaffold and about 1.5 million cells per scaffold, at times between about 0.5 million cells per scaffold and about 1 million cells per scaffold.

[0409] The method of the present discloses comprise inducing differentiation in the cells growth on the plurality of scaffolds and / or within the pores of the plurality of scaffolds.

[0410] The term differentiation of cells as used herein refers to process by which cells undergo changes, becoming specialized into various distinct cell types.

[0411] In the context of the present disclosure, cells undergo differentiation on at least one surface of the plurality of scaffolds and / or within the pores of the plurality of scaffolds. The time period at which the cells are allowed to differentiate in the bioreactor is denoted herein as differentiation phase. The differentiation phase is subsequent to the growth phase.

[0412] In some examples, the method comprises providing conditions that support differentiation of the cells. In some examples, the method comprises providing conditions that support differentiation of at least part of the cells. As appreciated, the conditions during the differentiation phase are optimized to suit the cells undergoing differentiation in the bioreactor and may vary depending for example, on the cell type into which the cells will differentiate.

[0413] In some examples, the conditions employed during the growth phase are the same as in the differentiation phase. In some examples, one or more of the temperature, pH, dissolved oxygen (DO), agitation. Osmolarity and gas exchange employed during the growth phase are the same as in the differentiation phase.

[0414] In some examples, the method comprises perfusing differentiation media through the basin.

[0415] As described herein, the cells grown on at least one surface of the plurality of scaffolds and / or within the pores of the of the plurality of scaffolds may differentiate into a large number of specificized differentiated cells. One of the conditions affecting the type of the specificized differentiated cells is the differentiation media.

[0416] Hence, the selection of cell media that is used during the differentiation phase may affect the type of specialized differentiated cells.

[0417] In some examples, the cell media used in the differentiation phase is a muscle differentiation media.

[0418] The term muscle differentiation medium as used herein refers to a cell culture medium used to promote differentiation into muscle cells, specifically mature muscle cells or myo tubes.

[0419] In some examples, the cell media used in the differentiation phase is an adipocyte differentiation media. The term adipocyte differentiation medium as used herein refers to a cell culture medium used to promote differentiation into adipocytes. In some examples, the adipocyte differentiation medium comprises one or more adipocyte differentiation induction agent. In some examples, the adipocyte differentiation induction agent includes one or more of insulin, dexamethasone or 3-Isobutyl-l -methylxanthine (IB MX). In some examples, the adipocyte differentiation medium comprises insulin and dexamethasone. In some examples, the method comprises inducing the cells toundergo differentiation into one or more of adipocytes, endothelial cells, hematopoietic cells, myocytes, hepatocytes, chondrocytes, osteocytes, fibroblast or any combination thereof.

[0420] In some examples, the method comprises inducing the cells to undergo differentiation into adipocytes.

[0421] In some examples, the method comprises inducing cells to undergo differentiation to form one or more of fat tissue, blood vessels, muscle tissue, liver tissue, cartilage tissue, bone tissue, connective tissue or any combination thereof.

[0422] In some examples, the method comprises inducing cells to undergo differentiation to form fat tissue.

[0423] The method comprises harvesting the porous three-dimensional scaffold structures with cells or tissue thereon.

[0424] The method comprises assessing at least one parameter to determine the optimal time or condition for cell harvesting. In some examples, the at least one parameter is at least one metabolite as described herein.

[0425] In some examples, the method comprises harvesting during cell growth and before differentiation begins.

[0426] In some examples, the method comprises harvesting undifferentiated cells.

[0427] In some examples, the method comprises harvesting the undifferentiated cells when glucose consumption rate reaches a stationary phase.

[0428] In some examples, the method comprises harvesting the undifferentiated cells when lactate formation rate reaches a stationary phase.

[0429] In some examples, the method comprises harvesting the undifferentiated cells when NH3 formation rate reaches a stationary phase.

[0430] In some examples, the method comprises harvesting the undifferentiated cells when LDH formation rate reaches a stationary phase. In some examples, the method comprises harvesting the undifferentiated cells between 1 day and 30 days after seeding, at times between 1 day and 20 days, at times between 1 day and 10 days.

[0431] In some examples, the method comprises harvesting the undifferentiated cells at 1 day after seeding, at times 2 days after seeding, at times 3 days, at times 4 days, at times 5 days, at times 6 days, at times 7 days, at times 8 days, at times 9 days, at times 10 days, at times 11 days, at times 12 days, at times 13 days, at times 14 days, at times 15 days, at times 16 days, at times 17 days, at times 18 days, at times 19 days, at times 20 days, at times 21 days, at times 22 days, at times 23 days, at times 24 days, at times 25 days, at times 26 days, at times 27 days, at times 28 days, at times 29 days, at times 30 days after seeding.

[0432] In some examples, the method comprises harvesting the undifferentiated cells at 7 days after seeding.

[0433] In some examples, the method comprises harvesting the differentiated cells. In some examples, the method comprises harvesting between 1 day to 30 days after initiating perfusion with differentiation media. In some examples, the method comprises harvesting after 1 day after initiating perfusion with differentiation media, at times after 2 days, at times 3 days, at times 4 days, at times 5 days, at times 6 days, at times 7 days, at times 8 days, at times 9 days, at times 10 days, at times 11 days, at times 12 days, at times 13 days, at times 14 days, at times 15 days, at times 16 days, at times 17 days, at times 18 days, at times 19 days, at times 20 days, at times 21 days, at times 22 days, at times 23 days, at times 24 days, at times 25 days, at times 26 days, at times 27 days, at times 28 days, at times 29 days, at times 30 days after initiating perfusion with differentiation media.

[0434] The method comprises collecting harvested porous three-dimensional scaffold structures with at least cells grown thereon.

[0435] In some examples, the method comprising collecting harvested porous three- dimensional scaffold structures with cell tissue grown thereon. In some examples, at least part of the cells is embedded within pores of the porous structures. In some examples, the cells are undifferentiated cells. In some other examples, the cells comprise different cells. In some examples, the cells comprise a single type of differentiated cells. In some examples, the cells comprise more than a single type of differentiated cells. In some examples, the cells comprise adipocyte cells. In some examples, the cells comprise muscle cells.

[0436] The porous three-dimensional scaffold structures with cell tissue grown thereon are collected by removing at least one end of the bioreactor.

[0437] The present disclosure may encompass a method in which cell seeding on the plurality of porous three-dimensional scaffold structures is done outside of the bioreactor followed by introducing the scaffolds having cells attached thereto are introduced into the packed bed bioreactor.

[0438] The present disclosure also encompasses a method in which the cells are seeded on the scaffolds externally to the bioreactor (i.e. not in the bioreactor). In other words, the cells are contacted with the scaffolds to allow seeding of cells thereon and the scaffolds with attached cells together are packed within the packed bed bioreactor.

[0439] In some aspects, the present disclosure provides a method comprising:

[0440] (i) introducing cell media comprising cells attached to a plurality of porous three-dimensional scaffold structures into a packed bed bioreactor comprising a basin having a first end, a second end and a wall extending between the first end and the second end defining an internal space, the bioreactor further including at least one fluid inlet at the first end and at least one fluid outlet; wherein the internal space is configured to hold the plurality of porous three-dimensional scaffold structures; wherein the plurality of porous three-dimensional scaffold structures comprises edible material; wherein the three-dimensional scaffold structures are packed within the basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of the porous three-dimensional scaffold structures within the internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet;

[0441] (ii) providing conditions that support growth and / or differentiation of the cells on the porous three-dimensional scaffold structures; and

[0442] (iii) harvesting the porous three-dimensional scaffold structures with cells thereon.

[0443] The present disclosure also provides in accordance with some other aspects an edible cultured cell mass comprising plurality of porous three-dimensional scaffold structures that includes edible material and at least cells carried by the edible material, by three-dimensional scaffold structures being distributed within by mass such that each three- dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure.

[0444] As described herein, the porous three-dimensional scaffold structures is suitable for cell growth and / or cell differentiation. In some examples, the plurality of three-dimensional scaffold structures comprises, adsorbed thereon, any one or combination of undifferentiated cells, differentiated cells, extracellular matrix protein and cell secreted metabolites.

[0445] In some examples, the edible cultured cell mass is characterized by a protein content of between about 0.5% and about 20% wet weight out of a total wet weight of said mass. In some examples, the edible cultured cell mass is characterized by a protein content of about 0.5%, at times about 1%, at times about 2%, at times about 3%, at times about 4%, at times about 5%, at times about 6%, at times about 7%, at times about 8%, at times about 9%, at times about 10%, at times about 11%, at times about 12%, at times about 13%, at times about 14%, at times about 15%, at times about 16%, at times about 17%, at times about 18%, at times about 19%, at times about 20% wet weight out of a total wet weight of said mass.

[0446] In some examples, the edible cultured cell mass is characterized by a lipid content of between about 0.1% and about 40% wet weight out of a total wet weight of said mass, at times between about 0.1% and about 30%, at times between about 0.3% and about 30%, at times between about 0.4% and about 25%, at times between about 0.5% and about 20% wet weight out of a total wet weight of said mass.

[0447] In some examples, the edible cultured cell mass is characterized by a lipid content of about 0.1%, at times about 0.3%, at times about 0.5%, at times about 0.7%, at times about 1%, at times about 3%, at times about 5%, at times about 7%, at times about 10%, at times about 13%, at times about 15%, at times about 17%, at times about 20%, at times about 23%, at times about 25%, at times about 27%, at times about 30%, at times about 33%, at times about 35%, at times about 37%, at times about 40% wet weight out of a total wet weight of said mass.

[0448] In some examples, the edible cultured cell mass is characterized by a lipid to protein weight ratio of between about 0.005 and about 80, at times between about 0.025 and about 40, at times between about 0.1 and about 1. In some examples, the edible cultured cell mass is characterized by a lipid to protein weight ratio of about 0.005, about 0.01, about 0.025, about 0.05, about 0.1, about 0.5, about 0.7, about 1, about 3, about 5, about 7, about 10, about 13, about 15, about 17, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 70, about 80.

[0449] In some examples, the edible cultured cell mass is characterized by a collagen to protein weight ratio of between about 1% and about 50%. In some examples, the edible cultured cell mass is characterized by a collagen to protein weight ratio of about 1%, at times of about 2%, at times of about 5%, at times of about 7%, at times of about 10%, at times of about 15%, at times of about 20%, at times of about 25%, at times of about 30%, at times of about 35%, at times of about 40%, at times of about 45%, at times of about 50%.

[0450] As shown in Figure 12C, the tissue grown in the bioreactor can be used for preparation of a food product.

[0451] Hence, in accordance with some other aspects, it is provided a food product comprising an edible cultured cell mass comprising plurality of porous three-dimensional scaffold structures that includes edible material and at least cells carried on said edible material; said three-dimensional scaffold structures being distributed within said mass such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure.

[0452] The food product is a cultivated food product. In some examples, the food product is a cultivated meat product.

[0453] The present disclosure also provides a cultivating system comprising a packed bed bioreactor comprising a container having a first end, a second end and a wall extending between said first end and said second end defining an internal space, said bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein said internal space is filled with plurality of porous three-dimensional scaffold structures; wherein said plurality of porous three-dimensional scaffold structures comprises edible material, wherein said three-dimensional scaffold structures are packed within said basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of said porous three-dimensional scaffold structures within said internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet; a cell media reservoir in fluid communication with said internal space; an oxygen source in fluid communication with said internal space; a control module configured for controlling at least one parameter during operation of said bioreactor.

[0454] The cell media reservoir is used to supply cell culture medium into the at least one packed bioreactor.

[0455] In some examples, the cell media reservoir comprises cells.

[0456] In some examples, the cell media reservoir comprises growth medium. In some examples, the cell media reservoir comprises differentiation medium.

[0457] In some examples, the cell media reservoir comprises oxygen dissolved in the growth medium and / or the differentiation medium.

[0458] The cell media reservoir is configured to supply to the at least one packed bed bioreactor a cell culture medium, configured to support the seeding, growth and / or differentiation of cells and / or tissues and as described herein is in fluid communication with the internal space of the at least one bioreactor.

[0459] The term fluid communication refers to means allowing flow of fluid between two components, such as pipes, tubes and the like.

[0460] In some examples, the basin is positioned in an orientation to allow flow up of fluids from the first fluid inlet upwards, and through the packed bed.

[0461] In some examples, the cell culture medium is introduced via the first fluid inlet.

[0462] In some examples, the fluid outlet is located on the first end. In some other examples, the fluid outlet is located on the second end.

[0463] The cultivating system may comprise additional components. In some examples, the cultivating system comprising at least one port accommodating at least one sensor as described herein. In some examples, the control module is configured to receive data from the at least one sensor and operate the bioreactor based on the received data.

[0464] The cultivating system comprising at least one pump.

[0465] In some examples, the at least one pump is configured to control the flow rate of medium flowing through the bioreactor.

[0466] In some examples, the at least one pump is configured for directing flow of fluid from the cell media source and / or oxygen source into the internal space.

[0467] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. For example, a pH value described herein may be within ±0.5 or ±0.2 of the indicated value. It should be noted that various embodiments of this invention may be presented in a range format. 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. For example, description of a range such as from 1 to 6 or between 1 and 6 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 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6.

[0468] It is to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0469] Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise” , and variations such as “comprises” and “comprising” , will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0470] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, the term a bioreactor may include one or more bioreactors having the recited characteristics. For example, the term a scaffold includes one or more scaffolds, e.g. a plurlity of similar scaffolds or a plurlity of diffenret scaffolds having the recited characteristics.

[0471] The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

[0472] It should be noted that the various embodiments and examples detailed herein in connection with various aspects of the invention may be applicable to one or more aspects disclosed herein. It should be further noted that any embodiment described herein, for example, related to components of the food ingredient, may be applied separately or in various combinations. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0473] The phrases “in another embodiment” or any refence made to embodiment as used herein do not necessarily refer to different embodiment, although it may. Thus, various embodiments of the invention can be combined (from the same or from different aspects) without departing from the scope of the invention.

[0474] NON-LIMITING EXAMPLES

[0475] Example 1A: Preparation of different edible scaffolds

[0476] The following describes preparation of different scaffolds.

[0477] Decellidarized (DC) edible scaffolds

[0478] Edible scaffolds (carriers) were prepared by cutting different plants (including fruits, vegetable, roots and stems) into different (e.g., disc or hexagonal) shape scaffolds. The disc shape scaffolds had a diameter of 6 mm and thickness of ~1 mm, the hexagonal shape scaffolds had a width of 6mm, side length of 3.5mm and thickness of 1 mm or 0.5 mm.

[0479] The scaffolds were cut using a template-based machine, ensuring uniformity and reproducibility in scaffold geometry.

[0480] Next, a de-cellularization step was performed as follows scaffolds were incubated with SDS (sodium dodecyl sulfate, #75746, Sigma) solution, for 48 hours at room temperature while gently shaken on an orbital shaker. Subsequently, the scaffolds were washed 3-5 times in deionized water (DW) and then incubated in a CaCh solution (#C1016, Sigma) for 24 hours at room temperature, using an orbital shaker and washed with DW. The cleaned scaffolds were frozen at -20°C for 24 hours before being transferred to a deep freezer (-80°C) for at least over-night incubation. Finally, scaffolds were freeze-dried for 48 hours and stored in a desiccator chamber until use.

[0481] Textured vegetable protein (TVP) TVP, TSP and TPP sourced from “Jinan DG” company, China. TVP sheets (~5mm thick) were precisely cut to the desired dimensions, for example 1cm x 1cm. Prior cell seeding, scaffolds were sterilized by autoclave and then underwent a thorough washing process x3 times with PBS.

[0482] Gelatin-based scaffolds

[0483] Various Gelatin-based scaffolds were purchased from Gelatex Technologies, Estonia, including #0601RN-SH and #compressed 0601RN-SH (were used as the scaffolds in Figures 2H and 21, respectively). The sheets were cut to the exact dimensions needed for the experiment (e.g., to a disc or hexagonal shape). Prior cell seeding, scaffolds were sterilized by autoclave and then underwent a thorough washing process x3 times with PBS.

[0484] Example IB: Characterization of the scaffolds’ water absorption capacity

[0485] To characterize the water absorption capacity of the scaffolds and the porosity percentage, the dry and wet masses of the scaffolds were weighed. Water retention capacity and scaffold porosity were calculated.

[0486] Results

[0487] Example of physical characteristics of decellularized-plant hexagonal scaffold:

[0488] The resulting edible scaffold had a water retention capacity of -97%, and porosity of 63% This was determined using the following equations:

[0489] Scaffold Wet weight- scaffold Dry weight

[0490] Scaffold’s retention capacity =

[0491] Scaffold Wet weight

[0492] Accordingly, the wet weight was ~20 mg / scaffold and the dry weight was ~0.6mg / scaffold, providing a water retention capacity of -97% (by weight).

[0493] Scaffold Wet weight- scaffold Dry weight

[0494] Scaffold pore volume _ Water density _

[0495] Scaffold’s porosity =

[0496] Scaffold total volume Scaffold total volume o.ozgr- o.oooegr igr / cm30.0194cm3

[0497] = 0.63 x 100 = 63%

[0498] 0.306 cm30.306 cm3

[0499] The calculated scaffold total volume is 0.306cm3(hexagonal of 6 mm width x 1mm thick), providing porosity of 63%. Example 2: Growth conditions of cells in edible packed bed (EPB) bioreactor

[0500] Example 2A: The effect of different scaffold materials on cell growth

[0501] Materials and Methods:

[0502] Edible scaffolds of different types were prepared as described above, in Example 1. 3-5 days prior seeding, the scaffolds underwent plasma treatment as described below, in Example 2C. Next, scaffolds were sterilized using dry autoclave followed by attachment solution coating step as described below in Example 2C.

[0503] Poultry fibroblast cells (Figures 2B-2G) or poultry adult stem cells (pASCs, isolation described in Example 4) (Figures 2H and 21) were seeded on the different scaffolds as follows: cell pellets were suspended in growth medium (GM; -Cell growth medium: DMEM / F-12 (HIMEDIA, Cat# - AT1184A), 10% FBS (HyClone, Cat# - SH30071.03), 1% glutamax (Gibco, Cat# - 35050-038), and 0.1% Gentamycin (Gibco, Cat# - 15750). Cell suspensions were added to a bottle with a specific number of scaffolds (2.5 million cells per 25 scaffolds= seeding density of O.lM / scaffold) and gently shaken on an orbital shaker at 39°C for 3-4 hours. After 24 hr, growth medium was replaced and cells were allowed to grow on an orbital shaker at 38°C for 7-10 days, changing growth medium every 2-3 days.

[0504] To evaluate cell growth Alamar Blue assay was performed - samples from the cultured mass were placed in growth medium with 10% alamar blue solution (Biorad, Cat# - BUF012B), followed by incubation in 39°C for 3 hours. The medium was then sampled, and signal was collected using a plate reader following the company provided protocol. The signal intensity is correlated to the number of cells. To display the cell growth rate, the signal at each time point was normalized to day 1.

[0505] Staining and imaging- cultured scaffolds were fixated in 4% paraformaldehyde (PFA) for 20-30 min and washed x3 with PBS. Then, scaffolds were stained for 4', 6- diamidino-2-phenylindole; Dapi (Sigma, Cat# D9542) (1 : 1000) and for Phalloidin (Sigma, Cat# P5282) (1:200) in PBS, on an orbital shaker at room temperature for 1 hr. Scaffolds were then imaged via LSM700 confocal microscope. Results:

[0506] The ability of different scaffolds to support cell growth was tested by growing cells on different scaffolds, including different plant-based scaffolds as compared to scaffold from an animal source, i.e. gelatin-based scaffolds.

[0507] As shown in Figure 2A, all the tested plant-based scaffolds supported cells growth with time.

[0508] Figures 2B-2I show confocal microscopy images of cells stained with Dapi and F- actin, hence providing information on cell’s nuclei and filamentous actin in the cytoskeleton.

[0509] The results show that all tested scaffolds including those prepared from fungi, decellularized vegetable root and decellularized plant stem, decellularized fruit, commercial TVP, commercial TSP and gelatin-based scaffolds supported cellular growth and proliferation indicating the versatility of the platform. These results demonstrate that different types of edible scaffolds and specifically plant-based scaffold are compatible for cell growth in EPB bioreactor.

[0510] Example 2B: The effect of different scaffold geometry and size on cell growth

[0511] Edible scaffolds of different geometries were prepared as described above in Example 1, and the bioreactor was packed with the plasma treated edible scaffolds.

[0512] The bioreactor was sterilized using dry autoclave, while the pH and dissolved oxygen (DO) sensors were autoclaved separately, and then placed aseptically in the bioreactor. The DO sensor was calibrated, and the bioreactor was filled with attachment solution (-Fibronectin attachment solution; Sartorius, Cat# - 05-750-1H) to allow attachment of cells to the edible carriers. Primary poultry adult stem cells (pASCs) (isolation described in Example 4), suspended in cell growth medium (GM; described in Example 2A) were added to the bioreactor, cells were attached to the scaffolds and allowed to grow in this medium for 7 days, while fresh medium was supplied to the system continuously via perfusion.

[0513] The growth medium was sampled daily, and glucose concentration was checked. Glucose consumption rate (GCR) between two samples was calculated by:

[0514] Q - Medium perfusion rate

[0515] V- Bioreactor volume

[0516] Gin - Glucose concentration in fresh medium

[0517] Go - Glucose concentration in first sample

[0518] Gf - Glucose concentration in second sample

[0519] At - time difference between two samples

[0520] BCA total protein analysis (indicative of protein content in cultivated product,) - was conducted using Pierce BCA Protein Assay Kit (ThermoFisher scientific, Cat# 23225) according to manufacture manual. Briefly the samples were exposed to lysis buffer and homogenized by sonication. Samples were then centrifuge and lysate was collected and mixed with BSA working reagent, followed by 30 minutes incubation, signal was then collected using a standard plate reader.

[0521] Results:

[0522] Cells growth was tested in different scaffold having different geometries and sizes, including hexagonal shape and circular shape (labeled as “Hex” and “Disc” in Figures 3A- 3C).

[0523] Figure 3A shows the thickness of the scaffold as measured from the top and bottom. As can be seen, two hexagonal scaffolds were tested with different thickness of 1mm and 0.5mm and one disc shape scaffold with a thickness of 1.3 mm.

[0524] As can be seen from Figure 3B, the rate of glucose consumption rate (GCR) was similar in the first days of cell growth on the different scaffold, with a slight increase in GCR observed after 7 days of cell growth in the hexagonal scaffold having a thickness of 0.5m.

[0525] As can be seen in Figure 3C, showing soluble protein concentration. It should be noted that GCR and the amount of the soluble protein concentration measured on all scaffolds were comparable.

[0526] These results demonstrate that different geometries and sizes of edible scaffolds were found to compatible for cell growth in EPB bioreactor.

[0527] Example 2C: The effect of plasma treatment of scaffolds on cell growth

[0528] Methods

[0529] To evaluate plasma treatment ability to expose the cellulose construct in decellularized plant material, edible scaffolds were prepared as described above, in Example 1.

[0530] The edible scaffolds were treated with plasma device- Vaculab, Tantec (2 mbar for 150 sec). Both plasma-treated and non-treated scaffolds were then immersed in 12 pM CBD-GFP solution, provided by BioBetter. Samples were incubated in 4°C for 3 hours, followed by two washes with PBS. Samples were than imaged for GFP via LSM700 confocal microscope.

[0531] To evaluate plasma treatment effect on cell growth and protein production on decellularized plant scaffold, both plasma-treated and non-treated scaffolds were incubated in fibronectin attachment solution (Sartorius, Cat# - 05-750- 1H, 1:50 dilution) in room temperature for 1 hr. pASCs were then seeded on the carriers and grown in cell growth medium in 37°C incubator for a duration of 21 days, medium was refreshed every 2-3 days.

[0532] To evaluate cell growth Alamar Blue assay was performed (as described in Example 2A).

[0533] To evaluate soluble protein concentration BCA total protein analysis was conducted as described in Example 2B.

[0534] Samples were imaged for GFP via LSM700 confocal microscope.

[0535] Results

[0536] The effect of plasma treatment on edible scaffold is shown in Figures 4A and 4B. As can be seen in these figures, a relatively low fluorescence intensity was observed in untreated scaffold (Figure 4A), whereas a strong fluorescence intensity was observed in plasma treated decellularized scaffold (Figure 4B).

[0537] The results suggest that plasma treatment expose cellulose structures and hence may be useful for cell attachment.

[0538] The effect of plasma treatment on cell growth is shown in Figure 4C. As can be seen, an increased cell growth was observed in the plasma treated scaffold. In addition to cell growth, an increase in the amount of soluble protein was observed in cells grown on plasma treated scaffolds as shown in Figure 4D.

[0539] Example 2D: The effect of atachment solutions on cell growth

[0540] Methods

[0541] To evaluate the effect of different attachment solutions on cell growth on decellularized plant scaffold, scaffolds were prepared, and plasma treated as describe above. Scaffolds were then incubated separately with one of the following:

[0542] 1. Attachment solution - Fibronectin attachment solution (O.Olmg / ml)

[0543] 2. Spent medium - waste medium of cultured cells, consisting of proteins and materials secreted by the cells.

[0544] 3. W / O attachment - only PBS pASCs were then seeded on the carriers and grown in cell growth medium in 37 °C incubator for a duration of 7 days. Viable cell number on scaffolds was evaluated on day 1 , 4 and 7 via Alamar blue assay as described in example 2A.

[0545] Samples were stained for Dapi and Phalloidin as described in Example 2A.

[0546] Results

[0547] As can be seen in Figure 5A, an increase in cell growth was shown in the two tested attachment solutions as compared to the cells grown without any solution.

[0548] Figures 5B-5D show cell growth and proliferation. As can be seen in these figures, treatment with solution (attachment, spent media and PBS) may be beneficial for cellular growth and proliferation. Example 3: Growth of different cells on edible scaffolds

[0549] Materials and Methods:

[0550] Poultry Adult stem cells (pASCs) Cells were isolated from a 26-day-old Pekin duck embryo. The tissue was thoroughly washed with a biological buffer, cut into small pieces, and enzymatically digested. The solution was then strained through 100 pm mesh, cells were centrifuged at 300 g for 10 min. Cell pellet was resuspended in GM and were subsequently seeded in a cell culture plate. Mycoplasma testing was conducted throughout the isolation process.

[0551] Bovine Adult stem cells (bASCs): cells were isolated from adult bovine tissue. The tissue was thoroughly washed with water, cut and placed in a sterile bag containing antibacterial and antifungal agents. The isolation process was carried out aseptically in a biological safety cabinet. The tissue was then cut, washed with a biological buffer, and digested to release the cells. The isolated cells were seeded in a tissue culture plate, with mycoplasma testing conducted at all stages of the isolation process. bASCs growth medium: DMEM- High glucose (ATCC, Cat# 30-2002), 10% FBS (Gibco, Cat# 19270- 106), 0.1 % Gentamicin (Gibco, Cat# - 15750).

[0552] Bovine Dermal Fibroblasts (BDFs): cells were purchased from ScienCell Research Laboratories and are isolated from fetal bovine skin. BDFs growth medium: Fibroblast Medium-2 (FM-2, Cat. #2331), 5% FBS FBS (Cat.#0025), 1% penicillin / streptomycin solution (P / S, Cat. #0503), 1% of Fibroblast Growth Supplement- 2 (FGS-2, Cat.#2382).

[0553] All cell types were cultured as previously described. After 7 days, the scaffolds were fixed with 4% paraformaldehyde (PFA) and stained for F-actin and nuclei using phalloidin and DAPI, respectively as described in Example 2A. The scaffolds were then visualized using a Zeiss LSM 700 confocal microscope.

[0554] Results:

[0555] As can be seen from Figures 6A-6H, various type of cells of different species were successfully grown on edible scaffolds. As can be seen, edible scaffolds were shown to be compatible with poultry and bovine cells such as adult stem cells (ASCs) and fibroblasts. Example 4: Characteristics of cell growth on edible scaffold

[0556] Methods

[0557] To demonstrate cell growth inside the edible packed bed, edible scaffolds of different geometries were prepared as described above, in Example 1 , and the bioreactor was packed with the plasma treated edible scaffolds. The bioreactor was sterilized using dry autoclave, while the pH and dissolved oxygen (DO) sensors were autoclaved separately, and then placed aseptically in the bioreactor. The DO sensor was calibrated, and the bioreactor was filled with attachment solution to allow attachment of cells to the edible carriers.

[0558] Chicken embryonic fibroblast cells (CEFs; purchased from ATCC, Cat# CRL - 3586) suspended in cell growth medium were added to the bioreactor, cells were attached to the scaffolds and allowed to grow in this medium for 10 days, while fresh medium was supplied to the system continuously via perfusion.

[0559] After biomass harvest, scaffolds were separated and underwent through fixation in 4% PFA and stained for Dapi and phalloidin as described in Example 2A. Scaffolds were then imaged via LSM700 confocal microscope from both sides of the scaffold to demonstrate cell mass formation on both sides of the edible scaffold.

[0560] Additionally, biomass was photographed to capture ECM formation between the scaffolds.

[0561] To demonstrate cell growth and tissue formation within the scaffold pores, duck embryonic fibroblasts (DEF; purchased from ATCC, Cat# - CCL-141) were seeded to a decellularized plant scaffold at cell density of 20k cells / scaffold. Cells were cultured in DEF growth medium (EMEM (ATCC, Cat# - 30-2003), 10% FBS (Gibco, Cat# - 19270- 106), and 0.1% Gentamycin (Gibco, Cat# - 15750)) for 14 days. Formed constructs were then fixated in 4% PFA, followed by paraffin sectioning (5pm sections) and Sirius red staining. Sections were then imaged using Nikon inverted microscope eclipse Ts2.

[0562] Results

[0563] The growth and distribution of CEFs on the scaffolds was tested. Figures 7A and7B are confocal microscopy images of cells stained with Dapi and F-actin, taken from both sides of the scaffold (i.e. opposite sides). As can be seen in these figures, a homogenous distribution of the cells was observed on all surfaces of the scaffold.

[0564] Figures 7C and 7D show biomass obtained after growth of CEFs showing growth of these cells within the scaffolds.

[0565] Figures 7E-7N show cross sections of DEF throughout the tested scaffold, stained with Sirius-red staining. As can be seen from these figures, cell distribution was observed within the scaffold at all tested depth.

[0566] The results suggest that the scaffold supports three-dimensional (3-D) growth of the cells.

[0567] Example 5: Growth of cell types in edible packed bed bioreactor

[0568] For fat differentiation, cells were induced with Adipocyte differentiation medium: DMEM / F-12 (Sartorius, Cat# - 01-170-1A), 5% FBS (Sigma, Cat# - F7524), 1% glutamax (Gibco, Cat# - 35050-038), 0.1% Gentamycin (Gibco, Cat# - 15750) ), Insulin 20ug / ml (Merck, Cat# - 16634), Deaxmesone luM, Oleic acids 40ug / ml (Sigma, Cat# - 01383), Linoleic acids 40ug / ml (Sigma, Cat# - L1012), D-Glucose, 4.5mg / ml (Sigma, Cat# - G8769).

[0569] Edible scaffolds were plasma treated using the vacuum instrument, and the bioreactor was packed with the plasma treated edible scaffolds (for example as shown in Figure 5A). The bioreactor was sterilized using dry autoclave, while the pH and dissolved oxygen (DO) sensors were autoclaved separately, and then placed aseptically in the bioreactor. The DO sensor was calibrated, and the bioreactor was filled with attachment solution to allow attachment of cells to the edible carriers.

[0570] Cells suspended in cell growth medium were added to the bioreactor, cell were attached to the scaffolds and allowed to grow in this medium for 7 days. On the 7thday, the medium was replaced to the adipocyte differentiation medium, allowing the cells to undergo differentiation.

[0571] During these days of cell growth and differentiation in the packed bed bioreactor, the medium (either growth medium or differentiation medium) was sampled daily, and concentrations of glucose, lactate, ammonia, glutamine, and lactate dehydrogenase were quantified using Cedex Bio analyzer. Metabolites consumption or formation rates were calculated and used to assess cell culture development (for growth and / or differentiation).

[0572] Control experiments included growth of cells in Erlenmeyer (“suspension”) and in wells (“direct wells”). Specifically, edible scaffolds were placed in an Erlenmeyer with attachment medium, after which, the attachment medium was replaced with growth medium including cells and the cells were allowed to grow for 7 days after which the cells were characterized as the cells grown in the packed bed. In addition, edible scaffold were placed in wells, such that each scaffold was placed in a well and attachment medium was added to each well. Then, the attachment medium was replaced with growth medium including cells and the cells were allowed to grow for 7 days after which the cells were characterized as the cells grown in the packed bed.

[0573] Results

[0574] During cell growth and differentiation, different parameters were monitored using increased GCR assay. Figures 8A-8D provide graphs of glucose consumption (Figure 8A), NH3 formation (Figure 8B), Lactate formation rate (Figure 8C) and Lactate dehydrogenase (LDH) formation (Figure 8D). The data was collected almost daily during a period of 21 days. The increase in all parameters during the growth period of the cells (days 0-7), support the conclusion that the edible scaffold supported cell growth in the EPB bioreactor. On the 21thday, the tissue was harvested.

[0575] Example 6: Tissue harvesting

[0576] Materials and Methods:

[0577] -Alamar Blue assay (indicative of cell density) -samples from the cultured mass were placed in growth media with 10% alamar blue solution (Biorad, Cat# - BUF012B), followed by incubation in 37°C for 3 hours. The media was then sampled, and signal was collected using a plate reader following the company provided protocol.

[0578] -Collagen purple staining (indicative of ECM formation and deposition) - the edible carriers were fixed using 4% PFA, followed by paraffin embedding. Transverse sequential paraffin sections (every 200 pm) of the scaffolds were stained with Sirius Red for Collagen staining.

[0579] -LipidTox staining (indicative of triglyceride production by differentiated fat cells) - tissue was fixed using 4% PFA, washing three time with PBS, and then stained with HCS LipidTOX (ThermoFisher scientific, Cat# H34475) according to the manufacture directions.

[0580] -BCA total protein analysis (indicative of protein content in cultivated product) - was conducted by using Pierce BCA Protein Assay Kit (ThermoFisher scientific, Cat# - 23225) according to manufacture manual. Briefly the samples were exposed to lysis buffer and homogenized by sonication. Samples were then centrifuge and lysate was collected and mixed with BSA working reagent, followed by 30 minutes incubation, signal was then collected using a standard plate reader.

[0581] -Triglyceride quantification (indicative of triglycerides in cultivated product) - was conducted by using Triglyceride Colorimetric Assay Kit (Cayman, Cat# - 10010303) according to manufacture manual. Briefly, the samples were exposed to lysis buffer and homogenized by sonication. Samples were then centrifuge and lysate was collected and mixed with triglycerides enzyme mix. colorimetric signal was then collected using a standard plate reader.

[0582] Results

[0583] Characterization of the cultivated tissue was done at the end of the growth phase (i.e. day 7) and at the end of the differentiation phase (i.e. day 21). Specifically, collagen stating (performed to evaluate ECM deposition) and total protein analysis were characterized at the end of the growth phase, whereas lipid staining, and TG analysis were characterized at the end of the differentiation phase.

[0584] To collect the edible scaffold with the tissue growth on them, the control loops were stopped, and the sensors, tubing and rotator were disconnected from the vessel. The vessel was placed in an aseptic environment and opened, the packed bed opened, and edible scaffold with cell culture biomass (tissue) were taken out to later analysis and integration into final hybrid product.

[0585] Characterization of the tissue in the harvested material is shown in Figures 9A-9H. Figure 9A shows nucleus and actin staining from tissue harvested after the growth phase.

[0586] Figures 9B and 9F show collagen staining (red purple) and collagen analysis and Figures 9D and 9E show protein analysis, respectively, from the tissue harvested after the growth phase.

[0587] As can be seen from Figure 9A, during the growth of cells in the bioreactor, the cells grew and proliferated and as shown in Figure 9B, there was growth of collagen suggesting development of extracellular matrix.

[0588] Protein content analysis including collagen (Figures 9D-9F) showed increased protein level in cells grown in packed bed configuration compared to cells grown in suspension in Erlenmeyer or cell pellets (control).

[0589] Figure 9C shows LipidTOX staining (green) and Figures 9G and 9H show triglyceride (TG) analysis, respectively, from the tissue harvested after the differentiation phase (after 21 days).

[0590] As can be seen from Figure 9C, during the differentiation, there was formation of lipids as shown in formation of lipid droplets.

[0591] As can be seen in Figures 9G and 9H, an increased lipid content was shown in tissue undergoing differentiation in packed bed configuration compared to cells undergoing differentiation in suspension in an Erlenmeyer or cell pellets. The results indicate that there was an advance differentiation to fat tissue.

[0592] In addition, to evaluate the homogeneity of the tissue growth in the packed bed configuration, scaffolds from different regions (top and bottom) from packed bed were sampled and product homogeneity analysis was conducted as follows:

[0593] The number of cells grown in the EPB was compared to the number of cells grown in wells. The number of cells was determined using the Alamar Blue assay. Figure 10A shows that when using the packed bed configuration, a significantly improved cell count was obtained as compared to cells seeded with "Direct" (cells directly injected into wells to the same scaffold) or in "Suspension" (cells attached to the same scaffold after suspended in the media covering the scaffold) In addition, as can be seen from Figure 10A, cell growth distributed uniformly within all packed bed as indicated in insignificant difference between the mass taken from the "Top" and the "Bottom" areas of the packed bed.

[0594] As shown in Figures 10B-10D, cell growth was comparable in all parts of the bioreactor.

[0595] Example 7: Muscle cell differentiation in EPB bioreactor

[0596] Methods:

[0597] To evaluate the compatibility of EPB platform for myogenic differentiation, poultry embryonic myoblasts (PEMs) cells (provided by ProFuse, Israel) were seeded on edible decellularized plasma-treated scaffolds (IM cells / scaffold). Cells were cultured in incubator at 37°C for 2 days with proliferation medium followed by either 2 days of myogenic differentiation medium or another 2 days with proliferation medium (Undifferentiated) (media provided by ProFuse).

[0598] Staining and imaging: After 4 days, scaffolds were fixed with 4% paraformaldehyde (PF A) and stained for Myosin heavy chain (MyHC) and DAPI. The scaffolds were then imaged using a Zeiss LSM 700 confocal microscope.

[0599] Western blot was used to detect the level of MyHC protein separated by SDS-PAGE. Protein extractions were run in SDS-PAGE gel. Followed by transfer to nitrocellulose membranes. Next, membranes were blocked with 5% skim milk and incubated with primary antibodies for 2-3 h at room temperature. After incubation with the primary antibody, membranes were washed 3 x 5 min. Membranes were then incubated with secondary antibodies for 1 h at room temperature, followed by 3 x 5 min washes. Bands were detected using gel scanner and densitometry was used to quantify bands intensity. MyHC levels were normalized to GAPDH bands.

[0600] Results:

[0601] Figures 11A-11B are images showing formation of muscle after differentiation of cells. As can be seen in Figure 11 A, elongated, fiber-like shapes of muscle cells (myotubes) were observed. In addition, figure 11B shows formation of multinucleated aligned myotubes during differentiation. These results indicate differentiation into muscle cells. Figure 11C show the presence of MHC protein only in the differentiated cells indicating differentiation into matured muscle fibers. Quantification of the data is shown in Figure 11D.

[0602] Example 8: Final product preparation

[0603] RESULTS

[0604] Figure 12A shows a representative packed bed bioreactor including the cellulose- based disc shaped scaffolds made of decellularized scaffold cut into a diameter of 6mm and thickness of 1 mm, as described above, before any treatment with medium and seeding of cells. Figure 12B is an image of the harvested scaffold material after growth of the adult stem cells as described above. The cell growth was confirmed by Alamar blue assay.

[0605] Figure 12C provides an image of the harvested cell-adhered scaffold structures after frying. For organoleptic properties, the fried product was tested by taster panel, which confirmed it had similar features as meat, in terms of appearance, texture, smell and taste.

[0606] As can be seen from Figure 13, an exemplary cultivating pilot system comprises a plurality of packed bed exemplary bioreactors (200), at least one fluid inlet (220), at the first end (240) and at least one fluid outlet (260).

[0607] Example 9: Sensory effect of cultivated meat product

[0608] In order to assess the sensory effect of cultivated meat product in EPB, this cultivated meat product was compared to cell slurry mix.

[0609] Methods:

[0610] Samples, composed of different weight percentages were prepared as follows:

[0611] 1. 96% growth medium and 4% scaffolds (denoted 0% Cultivated).

[0612] 2. 89.2% growth medium, 4% scaffolds, 6.8% cell slurry (Chicken embryonic fibroblasts), (denoted 6.8% Cultivated).

[0613] 3. 46% growth medium, 4% scaffolds, 50% cell slurry (denoted 50% Cultivated).

[0614] After mixing each sample until it was homogeneous, each sample was pan-fried for the precise amount of time and heat. All samples weighted the same (3gr). In addition, a sample of tissue that was grown in the EBP was pan-fried under the same conditions. This sample was characterized by a 6.8% of cells (grown on and within the scaffold).

[0615] Each fried sample inserted to opaque vile with random numbering for each sample for the test to be blind.

[0616] Group of 19 smellers was asked (each smeller separately) to rate the chicken smell intensity of each sample in the next scale: very low, low, mid, high, very high.

[0617] After each smeller finished rating the samples, all the samples reheated in the viles for the next smeller.

[0618] Each smeller was asked to rate the sample in comparison to fried chicken reference.

[0619] Results:

[0620] Figure 14 shows the average score of each sample, and the scattering of every ranking. As can be seen, cultivated meat product that was prepared in the EBP (with 6.8% cells) is characterized by a higher organoleptic effect as compared to a hybrid product with the same % of cell slurry.

[0621] In addition, cultivated meat product that was prepared in the EBP (with 6.8% cells) is characterized by the same organoleptic effect of hybrid product composed of 50% cell slurry.

[0622] These results demonstrate that cell and tissue growth in the EBP is advantageous, as it provides desirable organoleptic properties with a low cell density. This suggests that the increased productivity of cells and tissue production in the EBP is associated with the release of volatile compounds that mimic the sensory characteristics of traditional food products.

Claims

CLAIMS:

1. A packed bed bioreactor comprising a basin having a first end, a second end and a wall extending between said first end and said second end defining an internal space, said bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein said internal space is filled with plurality of porous three-dimensional scaffold structures; wherein said plurality of porous three-dimensional scaffold structures comprises edible material; wherein said three-dimensional scaffold structures are packed within said basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of said porous three-dimensional scaffold structures within said internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet.

2. The packed bed bioreactor of claim 1 , wherein said edible material comprises plant material.

3. The packed bed bioreactor of claim 2, wherein said plant material is a fruit or vegetable derived material.

4. The packed bed bioreactor of any one of claims 1 to 3, wherein each of said porous three-dimensional scaffold structures is characterized by one or more of:(i) comprise at least one dimension having a size within a range of about 0.5mm and about 500mm,(ii) comprise pores having a diameter of between about 20pm and about 800pm,(iii) porosity ranging from between about 50% and about 99%,(iv) having a shape selected from the group consisting of sheets, fibers, beads, flaks, discs, spheres, cylinders, rings, polygons and star shapes.

5. The packed bed reactor of any one of claims 1 to 4, comprising at least one type of three-dimensional scaffold structures.

6. The packed bed reactor of any one of claims 1 to 4, comprising at least two different types of three-dimensional scaffold structures.

7. The packed bed reactor of claim 6, wherein said at least two types of three- dimensional scaffold structures are different in at least one of scaffold material, scaffold size, scaffold shape, scaffold stiffness, scaffold elasticity, scaffold average pore size, scaffold porosity, scaffold color, scaffold texture or a combination thereof.

8. The packed bed reactor of any one of claims 1 to 7, wherein said plurality of scaffold structures is arranged in a homogenous mixture distribution.

9. The packed bed reactor of any one of claims 1 to 7, wherein said plurality of scaffold structures is arranged in a non-homogenous organized distribution.

10. The packed bed reactor of any one of claims 1 to 9, wherein said porous three- dimensional scaffold structures are pre-conditioned to promote adherence of cells onto said scaffold structures.

11. The packed bed bioreactor of claim 10, wherein said pre-conditioning comprises any one of plasma treatment, electrostatic charging of surface of said porous three- dimensional scaffold structures; increasing surface energy of said porous three- dimensional scaffold structures, removing lignin from surface of said porous three- dimensional scaffold structures; exposing cellulose residues on surface of said porous three-dimensional scaffold structures.

12. The packed bed bioreactor of any one of claims 1 to 11, comprising at least one port for accommodating at least one sensor.

13. The packed bed reactor of claim 12, wherein said at least one sensor is selected to detect at least one parameter selected from the group consisting of pH, dissolved oxygen (DO), temperature capacitance, glucose, lactate, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH), biomass weight, biomass level in basin, foam.

14. The packed bed bioreactor of any one of claims 1 to 13, wherein said basin is positioned in an orientation to allow flow up of fluids from said fluid inlet upwards, and through the packed bed.

15. The packed bed bioreactor of any one of claims 1 to 14, for use with a source of culture media for growing cells within said basin.

16. A method comprising:(i) introducing cell media comprising cells into a packed bed bioreactor comprising a basin having a first end, a second end and a wall extending between said first end and said second end defining an internal space, said bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein said internal space is filled with plurality of porous three- dimensional scaffold structures; wherein said plurality of porous three-dimensional scaffold structures comprises edible material; wherein said three-dimensional scaffold structures are packed within said basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of said porous three-dimensional scaffold structures within said internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet;(ii) providing conditions that support growth of said cells on said porous three- dimensional scaffold structures; and(iii) harvesting said porous three-dimensional scaffold structures with cells thereon.

17. The method of claim 16, wherein said packed bed bioreactor is as defined in any one of claims 1 to 15.

18. The method of claim 16 or 17, comprising pre-conditioning said porous three- dimensional scaffold structures to promote adherence of cells onto said scaffold structures.

19. The method of claim 18, wherein said pre-conditioning comprises any one of plasma treatment, electrostatic charging of surface of said porous three-dimensional scaffold structures; increasing surface energy of said porous three-dimensional scaffold structures, removing lignin from surface of said porous three-dimensional scaffold structures; exposing cellulose residues on surface of said porous three-dimensional scaffold structures.

20. The method of any one of claims 16 to 19, comprising washing said porous three- dimensional scaffold structures with an attachment solution, said washing is subsequent to said pre-conditioning, if said pre-conditioning is performed.

21. The method of claim 20, wherein said attachment solution is selected from the group consisting of fresh media, PBS, fibronectin attachment solution, collagen attachment solution, laminin attachment solution, vitronectin attachment solution, elastin attachment solution poly-L-lysine attachment solution, gelatin attachment solution and spent attachment solution.

22. The method of any one of claims 16 to 21, wherein said cell media comprises a single cell type or more than one cell type.

23. The method of any one of claims 16 to 22, wherein said cell media comprises at least stem cells.

24. The method of any one of claims 16 to 23, wherein said cell media comprises at least cells selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, adult stem cells, mesenchymal stem cells, myoblasts, satellite muscle cells, fibroblasts, hepatoblasts, osteoblasts, chondroblasts, adipocytes, hepatocytes, osteocytes, chondrocytes, and any combination of same.

25. The method of any one of claims 16 to 24, wherein said conditions that support growth of said cells on said porous three-dimensional scaffold structures comprise (i) temperature within a range of between about 10°C and about 45°C,(ii) pH within a range of between about 6 and about 8.(iii) level of DO within a range of between about 10% and about 100%,(iv) agitation with a shear stress of between about O.ldyn / cm2and about 10dyn / cm2.

26. The method of any one of claims 16 to 25, wherein said conditions that support growth of said cells on said porous three-dimensional scaffold structure comprise conditions supportive of cell seeding and conditions supporting of cell growth.

27. The method of claim 26, wherein said conditions comprise flow velocity between about 1 cm / sec and 5cm / sec.

28. The method of claim 27, wherein agitation during said seeding comprises agitation with alternating velocities including a first rotational speed in a range of 10-50rpm and a second rotational speed between lOOrpm and 200rpm.

29. The method of claim 28, wherein said agitation at said first rotational speed is for a period of between 10 minutes and 20 minutes, and said agitation at said second rotational speed is for a period of less than 1 minute.

30. The method of any one of claims 27 to 29, wherein said agitation during said cell growth is at a rotational speed within a range of about lOOrpm and about 200rpm.

31. The method of any one of claims 16 to 30, comprising monitoring at least one parameter selected from the group consisting of pH, dissolved oxygen (DO), temperature capacitance, metabolites, biomass weight, biomass level in basin and foam.

32. The method of claim 31 , wherein said metabolites comprises at least one metabolite selected from the group consisting of glucose, lactate, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH).

33. The method of any one of claims 16 to 32, perfusing growth and / or differentiation media through said basin.

34. The method of any one of claims 16 to 33 comprising providing said conditions that support growth of said cells.

35. The method of any one of claims 16 to 34, comprising providing said conditions that support differentiation of at least part of said cells.

36. The method of any one of claims 16 to 35, wherein said cells are undifferentiated cells and harvesting takes place when glucose consumption rate reaches a stationary phase.

37. The method of claim 36, wherein said cells comprise differentiated cells and harvesting takes place between 1 to 30 days after initiating perfusion with differentiation media.

38. The method of any one of claims 16 to 37, comprising collecting harvested porous three-dimensional scaffold structures with at least cells grown thereon.

39. The method of any one of claims 16 to 38, comprising collecting harvested porous three-dimensional scaffold structures with cell tissue grown thereon.

40. The method of claim 39, wherein at least part of the cells are embedded within pores of the porous structures.

41. An edible cultured cell mass comprising plurality of porous three-dimensional scaffold structures that includes edible material and at least cells carried by said edible material, said three-dimensional scaffold structures being distributed within said mass such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure.

42. The edible cultured cell mass of claim 41, wherein said plurality of three- dimensional scaffold structures comprise, adsorbed thereon, any one or combination of undifferentiated cells, differentiated cells, extracellular matrix protein and cell secreted metabolites.

43. The edible cultured cell mass of claim 41 or 42, being characterized by at least one of the following: protein content of between about 0.5-20% wet weight out of a total wet weight of said mass; lipid content of between about 0.1-40% wet weight out of a total wet weight of said mass; lipid to protein weight ratio of between about 0.1-1; collagen to protein weight ratio of between about 1-50%.

44. A food product comprising an edible cultured cell mass comprising plurality of porous three-dimensional scaffold structures that includes edible material and at least cellscarried on said edible material; said three-dimensional scaffold structures being distributed within said mass such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure.

45. The food product of claim 44, wherein said cultured cell mass is as defined in any one of claims 41 to 43.

46. A cultivating system comprising a packed bed bioreactor comprising a container having a first end, a second end and a wall extending between said first end and said second end defining an internal space, said bioreactor further including at least one fluid inlet at said first end and at least one fluid outlet; wherein said internal space is filled with plurality of porous three-dimensional scaffold structures; wherein said plurality of porous three-dimensional scaffold structures comprises edible material, wherein said three-dimensional scaffold structures are packed within said basin such that each three-dimensional scaffold structure is in contact with at least one neighboring three-dimensional scaffold structure; and wherein porosity and distribution of said porous three-dimensional scaffold structures within said internal space are selected to allow essentially uniform flow of fluid from said first fluid inlet to said fluid outlet; a cell media reservoir in fluid communication with said internal space; an oxygen source in fluid communication with said internal space; a control module configured for controlling at least one parameter during operation of said bioreactor.

47. The cultivating system of claim 46, wherein said basin is positioned in an orientation to allow flow up of fluids from said first fluid inlet upwards, and through the packed bed.

48. The cultivating system of claim 46 or 47, wherein said fluid outlet is located on said second end.

49. The cultivating system of any one of claims 46 to 48, comprising at least one port accommodating at least one sensor, and said control module is configured to receive data from said at least one sensor and operate said bioreactor based on said received data.

50. The cultivating system of any one of claims 46 to 49, comprising at least one pump for directing flow of fluid from said cell media source and / or oxygen source into said internal space.

51. The cultivating system of any one of claims 46 to 50, wherein said packed bed bioreactor is as defined in any one of claim 1 to 15.

52. The cultivating system of any one of claims 46 to 51 , configured for performing the method of any one of claims 36 to 40.

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