Bioreactors, methods and systems for producing cultured meat

Abstract

The present disclosure relates to a packed bed bioreactor, a method of use thereof, a cultured cell mass, and a food product comprising the cultured cell mass.

Description

Technical Field

[0001] This disclosure relates to the production of cultured meat. Background Technology

[0002] The following is a list of references considered relevant to the background art of the currently disclosed topic: -International application publication number WO2022038240 -International application publication number WO2022097139 -International application publication number WO2012140519 -US Application Publication No. US2021189329 The acknowledgment of the above references in this document should not be construed as implying that these references are in any way related to the patentability of the currently disclosed subject matter.

[0003] background WO20220382 describes an edible hollow fiber, a cartridge containing the hollow fiber, and a bioreactor. The hollow fiber comprises one or more materials selected from the group consisting of hydrophilic colloids 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. WO20220382 also describes a method for producing structured clean meat products using the hollow fiber, cartridge, and bioreactor, and the structured clean meat products produced by the method.

[0004] WO2022097139 describes the large-scale production of cultured cells and relates to a system comprising multiple scaffolds optionally arranged in a multilayer configuration. WO2022097139 also describes methods of using the system to produce cell and / or tissue cultures for a variety of uses, including the production of cultured food products, particularly cultured meat.

[0005] WO2012140519 describes the use of vibration to harvest cells grown in 3D culture. This method requires applying a force with sufficient amplitude, frequency, and duration to the cells attached to the 3D matrix to detach the cells from the matrix and flush the detached cells out of the matrix material. An apparatus for performing the disclosed method is also provided.

[0006] US2021189329 describes methods and systems for enhancing the production and / or secretion of extracellular vesicles from at least one three-dimensional porous scaffold on which a population of stem cells is cultured, the methods and systems utilizing various shear stress conditions on a variety of stem cells. Summary of the Invention

[0007] The subject matter disclosed in this invention is based on the discovery that cells and tissues can be effectively grown on edible scaffolds within a packed-bed bioreactor, thereby generating edible cell clusters that can form alternative food products.

[0008] Therefore, according to a first aspect of the subject matter disclosed in the present invention, a packed bed bioreactor is provided, the packed bed bioreactor including a cavity 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. The internal space is filled with multiple porous three-dimensional support structures; The plurality of porous three-dimensional scaffold structures contain edible materials; The three-dimensional scaffold structure is filled within the cavity such that each three-dimensional scaffold structure contacts at least one adjacent three-dimensional scaffold structure; and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet.

[0009] According to a second aspect of the subject matter disclosed in this invention, a method is provided, the method comprising: (i) Introducing a cell culture medium containing cells into a packed bed bioreactor, the packed bed bioreactor including a cavity 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. The internal space is filled with multiple porous three-dimensional support structures; The plurality of porous three-dimensional scaffold structures contain edible materials; The three-dimensional scaffold structure is filled within the cavity such that each three-dimensional scaffold structure contacts at least one adjacent three-dimensional scaffold structure; and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet. (ii) Providing conditions that support the growth and / or differentiation of the cells on the porous three-dimensional scaffold structure; and (iii) Harvest the porous three-dimensional scaffold structure containing cells thereon.

[0010] According to a third aspect of the subject matter disclosed in the present invention, an edible cultured cell mass is provided, the edible cultured cell mass comprising a plurality of porous three-dimensional scaffold structures, the scaffold structures comprising edible material and at least cells carried by said edible material (adsorbed thereon, embedded in pores, etc.).

[0011] According to a fourth aspect of the subject matter disclosed in the present invention, a food product is provided comprising an edible cultured cell cluster comprising a plurality of porous three-dimensional scaffold structures comprising edible material and at least cells carried by the edible material; the three-dimensional scaffold structures are distributed within the cell cluster such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure.

[0012] Implementation Plan Some embodiments of this disclosure will now be described in the following numbered paragraphs. The following description is intended to complement the general description above and is not intended to limit it in any way.

[0013] 1. A packed bed bioreactor, the packed bed bioreactor comprising a cavity 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 comprising at least one fluid inlet at the first end and at least one fluid outlet; The internal space is filled with multiple porous three-dimensional support structures; The plurality of porous three-dimensional scaffold structures contain edible materials; The three-dimensional scaffold structure is filled within the cavity such that each three-dimensional scaffold structure contacts at least one adjacent three-dimensional scaffold structure; and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet.

[0014] 2. A method, the method comprising: (i) Introducing a cell culture medium containing cells into a packed bed bioreactor, the packed bed bioreactor including a cavity 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. The internal space is filled with multiple porous three-dimensional support structures; The plurality of porous three-dimensional scaffold structures contain edible materials; The three-dimensional scaffold structure is filled within the cavity such that each three-dimensional scaffold structure contacts at least one adjacent three-dimensional scaffold structure; and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet. (ii) Providing conditions that support the growth of the cells on the porous three-dimensional scaffold structure; and (iii) Harvest the porous three-dimensional scaffold structure containing cells thereon.

[0015] 3. The packed bed bioreactor and / or method according to embodiment 1 or 2, wherein the edible material comprises plant material.

[0016] 4. A packed bed bioreactor and / or method according to any one of embodiments 1 to 3, wherein the edible material comprises at least insoluble dietary fiber.

[0017] 5. The packed bed bioreactor and / or method according to embodiment 4, wherein the insoluble dietary fiber comprises polysaccharides.

[0018] 6. The packed bed bioreactor and / or method according to any one of embodiments 1 to 5, wherein the edible material comprises a cellulose material.

[0019] 7. A packed bed bioreactor and / or method according to any one of embodiments 1 to 6, wherein the edible material comprises plant protein.

[0020] 8. The packed bed bioreactor and / or method according to embodiment 3, wherein the edible material is of fruit or vegetable origin.

[0021] 9. A packed bed bioreactor and / or method according to any one of embodiments 1 to 8, wherein the edible material comprises at least insoluble dietary fiber derived from fruit.

[0022] 10. The packed bed bioreactor and / or method according to embodiment 1, wherein the edible material comprises animal-derived material.

[0023] 11. The packed bed bioreactor and / or method according to embodiment 10, wherein the animal-derived material is selected from the group consisting of: decellularized tissue, collagen, gelatin, elastin, laminin, fibronectin, hyalin, and any combination thereof.

[0024] 12. The packed bed bioreactor and / or method according to any one of embodiments 1 to 11, wherein the edible material comprises a combination of plant material and animal-derived material.

[0025] 13. The packed bed bioreactor and / or method according to any one of embodiments 1 to 12, wherein each porous three-dimensional scaffold structure in the porous three-dimensional scaffold structure comprises at least one dimension with a size ranging from about 0.5 mm to about 500 mm.

[0026] 14. The packed bed bioreactor and / or method according to embodiment 13, wherein the porous three-dimensional scaffold structure has a first dimension and a second dimension that jointly define a plane, and a thickness perpendicular to the plane, the thickness being in the range of about 0.1 mm to about 2 mm.

[0027] 15. The packed bed bioreactor and / or method according to embodiment 14, wherein at least one of the first dimension and the second dimension is greater than the thickness.

[0028] 16. The packed bed bioreactor and / or method according to any one of embodiments 1 to 15, wherein the porous scaffold comprises a porous material having pores with a size between about 10 µm and about 800 µm. 17. The packed bed bioreactor and / or method according to any one of embodiments 1 to 16, wherein the porosity of the plurality of porous three-dimensional scaffold structures is in the range of about 50% to about 99%.

[0029] 18. A packed bed bioreactor and / or method according to any one of embodiments 1 to 17, wherein the plurality of porous three-dimensional scaffold structures are filled with a dry density in the range of about 1 mg / ml to 1000 mg / ml.

[0030] 19. A packed bed bioreactor and / or method according to any one of embodiments 1 to 18, wherein the plurality of porous three-dimensional scaffold structures are filled with a wet density in the range of about 0.1 g / ml to about 1.0 g / ml.

[0031] 20. A packed bed bioreactor and / or method according to any one of embodiments 1 to 19, wherein the three-dimensional scaffold structure has a shape selected from the group consisting of: sheet-like, fibrous, bead-like, flaky, disc-shaped, spherical, cylindrical, annular, polygonal, and star-shaped.

[0032] 21. The packed bed bioreactor and / or method according to any one of embodiments 1 to 20, wherein the packed bed bioreactor and / or method comprises at least one type of three-dimensional scaffold structure.

[0033] 22. The packed bed bioreactor and / or method according to any one of embodiments 1 to 20, wherein the packed bed bioreactor and / or method comprises at least two different types of three-dimensional scaffold structures.

[0034] 23. The packed bed bioreactor and / or method according to embodiment 22, wherein the at least two types of three-dimensional scaffold structures differ in at least one aspect 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.

[0035] 24. The packed bed bioreactor and / or method according to any one of embodiments 1 to 23, wherein the plurality of support structures are arranged in a uniformly mixed distribution.

[0036] 25. The packed bed bioreactor and / or method according to any one of embodiments 1 to 24, wherein the plurality of support structures are arranged in a non-uniform, ordered distribution.

[0037] 26. A packed bed bioreactor and / or method according to any one of embodiments 1 to 25, wherein the porous three-dimensional scaffold structure is pre-adjusted to promote cell adhesion to the scaffold structure.

[0038] 27. The packed bed bioreactor and / or method according to embodiment 26, wherein the pre-adjustment includes physical and / or chemical alterations to the surface of the porous three-dimensional scaffold structure.

[0039] 28. The packed bed bioreactor and / or method according to embodiment 26 or 27, wherein the pre-adjustment includes any one of the following: plasma treatment or electrostatic charging of the surface of the porous three-dimensional scaffold structure; increasing the surface energy of the porous three-dimensional scaffold structure; removing lignin from the surface of the porous three-dimensional scaffold structure; or exposing cellulose residues on the surface of the porous three-dimensional scaffold structure.

[0040] 29. A packed bed bioreactor and / or method according to any one of embodiments 28 to 27, wherein the pre-adjustment is determined by the level of cell adhesion to the porous three-dimensional scaffold structure under conditions containing positive charges and exposed cellulose binding sites.

[0041] 30. The packed bed bioreactor and / or method according to any one of embodiments 1 to 29, wherein the porous three-dimensional scaffold structure within the cavity is a freeze-dried structure.

[0042] 31. The packed bed bioreactor and / or method according to any one of embodiments 1 to 30, wherein the porous three-dimensional scaffold structure is sterilized.

[0043] 32. The packed bed bioreactor and / or method according to embodiment 32, wherein the porous three-dimensional scaffold structure is sterilized by any sterilization method selected from autoclaving, ultraviolet irradiation, radiation sterilization and dry heat sterilization.

[0044] 33. The packed bed bioreactor and / or method according to any one of embodiments 1 to 32, wherein the packed bed bioreactor and / or method includes a removable top plate configured to allow the introduction and / or removal of contents of the cavity.

[0045] 34. A packed bed bioreactor and / or method according to any one of embodiments 1 to 3332, wherein the packed bed bioreactor and / or method includes at least one port for accommodating at least one sensor.

[0046] 35. The packed bed bioreactor and / or method according to embodiment 34, wherein the 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, lactic acid, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH), biomass weight, biomass level in the chamber, and foam.

[0047] 36. The packed bed bioreactor and / or method according to any one of embodiments 1 to 35, wherein the packed bed bioreactor and / or method includes a mechanism configured to allow fluid to circulate within the internal space.

[0048] 37. The packed bed bioreactor and / or method according to embodiment 36, wherein the mechanism comprises at least one component selected from the group consisting of: impeller, peristaltic pump, diaphragm pump, vibrator, tilter and lifter.

[0049] 38. The packed bed bioreactor and / or method according to any one of embodiments 1 to 37, wherein the packed bed bioreactor and / or method includes at least one control loop.

[0050] 39. The packed bed bioreactor and / or method according to embodiment 38, wherein 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.

[0051] 40. A packed bed bioreactor and / or method according to any one of embodiments 1 to 39, wherein the orientation of the cavity is positioned to allow fluid to flow upward from the fluid inlet and through the packed bed.

[0052] 41. A packed bed bioreactor and / or method according to any one of embodiments 1 to 40, wherein the wall of the cavity includes at least one transparent segment configured to allow observation of the contents of the interior space.

[0053] 42. The packed bed bioreactor and / or method according to any one of embodiments 1 to 42, wherein the packed bed bioreactor and / or method is used in conjunction with a culture medium source for culturing cells within the cavity.

[0054] 43. The method according to any one of embodiments 1 to 42, the method comprising pre-adjusting the porous three-dimensional scaffold structure to promote cell adhesion to the scaffold structure.

[0055] 44. The method according to embodiment 43, wherein the pre-adjustment includes physical and / or chemical alterations to the surface of the porous three-dimensional scaffold structure.

[0056] 45. The method according to embodiment 43 or 44, wherein the pre-adjustment includes any one of the following: plasma treatment or electrostatic charging of the surface of the porous three-dimensional scaffold structure; increasing the surface energy of the porous three-dimensional scaffold structure; removing lignin from the surface of the porous three-dimensional scaffold structure; or exposing cellulose residues on the surface of the porous three-dimensional scaffold structure.

[0057] 46. ​​The method according to any one of embodiments 43 to 45, wherein the pre-adjustment is determined by the adhesion level of cells to the porous three-dimensional scaffold structure under conditions containing positive charges and exposed cellulose binding sites.

[0058] 47. The method according to any one of embodiments 43 to 46, wherein the method comprises lyophilizing the porous three-dimensional scaffold structure prior to contacting the structure with the cells.

[0059] 48. The method according to any one of embodiments 43 to 47, wherein the method includes sterilizing the porous three-dimensional scaffold structure prior to contacting the structure with the cells.

[0060] 49. The method according to embodiment 48, wherein the sterilization comprises any sterilization method selected from the group consisting of: autoclaving, ultraviolet irradiation, radiation sterilization, dry heat sterilization, and combinations thereof.

[0061] 50. The method according to any one of embodiments 43 to 49, the method comprising washing the porous three-dimensional scaffold structure with a solution, wherein the washing is performed after the pre-adjustment if the pre-adjustment has been performed.

[0062] 51. The method according to embodiment 50, wherein the solution is selected from the group consisting of: PBS, fibronectin attachment solution, collagen attachment solution, laminin attachment solution, fibronectin attachment solution, elastin attachment solution, poly-L-lysine attachment solution, gelatin attachment solution, and used attachment solution.

[0063] 52. The method according to any one of embodiments 43 to 51, the method comprising saturating the porous three-dimensional scaffold structure with a growth medium before introducing the cell culture medium and after the washing.

[0064] 53. The method according to any one of embodiments 43 to 52, wherein the cell culture medium comprises a single cell type or more than one cell type.

[0065] 54. The method according to any one of embodiments 43 to 53, wherein the cell culture medium contains at least stem cells.

[0066] 55. The method according to any one of embodiments 43 to 54, wherein the cell culture medium 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 myoblasts, fibroblasts, hepatocytes, osteoblasts, chondrocytes, adipocytes, hepatocytes, osteoblasts, chondrocytes, and any combination thereof.

[0067] 56. The method according to any one of embodiments 43 to 55, wherein the conditions supporting the growth of the cells on the porous three-dimensional scaffold structure include at least one condition selected from the group consisting of: temperature, pH, dissolved oxygen (DO) and agitation.

[0068] 57. The method according to any one of embodiments 43 to 56, wherein the conditions supporting the growth of the cells on the porous three-dimensional scaffold structure include a temperature in the range of about 20°C to about 45°C.

[0069] 58. The method according to any one of embodiments 43 to 57, wherein the conditions supporting the growth of the cells on the porous three-dimensional scaffold structure include a pH in the range of about 6 to about 8.

[0070] 59. The method according to any one of embodiments 43 to 58, wherein the conditions supporting the growth of the cells on the porous three-dimensional scaffold structure include a DO level in the range of about 10% to about 100%.

[0071] 60. The method according to any one of embodiments 43 to 59, wherein the conditions supporting the growth of the cells on the porous three-dimensional scaffold structure include having a density between about 0.1 dyn / cm 2 With approximately 10 dyn / cm 2 Stirring of shear stress between them.

[0072] 61. The method according to any one of embodiments 43 to 60, wherein the conditions supporting the growth of the cells on the porous three-dimensional scaffold structure include conditions supporting cell seeding and conditions supporting cell growth.

[0073] 62. The method according to embodiment 61, wherein the conditions include a flow rate between about 1 cm / s and 5 cm / s.

[0074] 63. The method according to embodiment 62, wherein the stirring during inoculation includes stirring with alternating speeds, the alternating speeds including a first rotational speed in the range of 10 rpm to 50 rpm and a second rotational speed between 100 rpm and 200 rpm.

[0075] 64. The method according to embodiment 63, wherein the stirring at the first rotational speed lasts for a period between 10 minutes and 20 minutes, and the stirring at the second rotational speed lasts for a period of less than 1 minute.

[0076] 65. The method according to any one of embodiments 63 to 64, wherein the stirring during cell growth is performed at a rotation speed in the range of about 100 rpm to about 200 rpm.

[0077] 66. The method according to any one of embodiments 43 to 65, the method comprising monitoring at least one parameter selected from the group consisting of: pH, dissolved oxygen (DO), temperature capacitance, metabolites, biomass weight, biomass level in the cavity, and foam.

[0078] 67. The method according to embodiment 66, wherein the metabolite comprises at least one metabolite selected from the group consisting of glucose, lactate, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH).

[0079] 68. The method according to any one of embodiments 43 to 67, wherein the cavity is perfused with growth medium and / or differentiation medium.

[0080] 69. The method according to any one of embodiments 43 to 68, the method comprising providing the conditions supporting the growth of the cells.

[0081] 70. The method according to any one of embodiments 43 to 69, the method comprising the conditions supporting the differentiation of at least a portion of the cells.

[0082] 71. The method according to any one of embodiments 43 to 70, wherein the cells are undifferentiated cells and are harvested when the glucose consumption rate reaches a plateau.

[0083] 72. The method according to embodiment 71, wherein the cells comprise differentiated cells and are harvested between 1 and 30 days after the start of perfusion with differentiation medium.

[0084] 73. The method according to any one of embodiments 43 to 72, the method comprising collecting harvested porous three-dimensional scaffold structures having at least growing cells thereon.

[0085] 74. The method according to any one of embodiments 43 to 73, the method comprising collecting harvested porous three-dimensional scaffold structures having growing cellular tissue thereon.

[0086] 75. The method according to embodiment 73 or 74, wherein at least a portion of the cell is embedded within the pores of the porous structure.

[0087] 76. An edible cultured cell cluster comprising a plurality of porous three-dimensional scaffold structures, each porous three-dimensional scaffold structure comprising edible material and at least cells carried by the edible material, the three-dimensional scaffold structures being distributed within the cell cluster such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure.

[0088] 77. The edible cultured cell cluster according to embodiment 76, wherein the plurality of three-dimensional scaffold structures comprise any one or a combination of undifferentiated cells, differentiated cells, extracellular matrix proteins, and cell-secreted metabolites adsorbed thereon.

[0089] 78. The edible cultured cell mass according to embodiment 76 or 77, characterized in that at least one of the following: - The protein content is between approximately 0.5% and 20% of the total wet weight of the cell cluster; - The lipid content is between approximately 0.1% and 40% of the total wet weight of the cell cluster; - The weight ratio of lipids to proteins is between approximately 0.1 and 1; - The weight ratio of collagen to protein is between approximately 1% and 50%.

[0090] 79. A food product comprising an edible cultured cell cluster, the edible cultured cell cluster comprising a plurality of porous three-dimensional scaffold structures, the porous three-dimensional scaffold structures comprising edible material and cells carried on at least the edible material; the three-dimensional scaffold structures are distributed within the cell cluster such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure.

[0091] 80. The food product according to embodiment 78, wherein the cultured cell cluster is as defined in any one of embodiments 76 to 78.

[0092] 81. A culture system, the culture system comprising - A packed bed bioreactor, the packed bed bioreactor comprising a container 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 comprising at least one fluid inlet and at least one fluid outlet at the first end; The internal space is filled with multiple porous three-dimensional support structures; The multiple porous three-dimensional scaffold structures mentioned above contain edible materials. The three-dimensional scaffold structure is filled within the cavity such that each three-dimensional scaffold structure contacts at least one adjacent three-dimensional scaffold structure; and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet. - A cell culture medium reservoir in fluid communication with the internal space; - An oxygen source in fluid communication with the interior space; - A control module configured to control at least one parameter during operation of the bioreactor.

[0093] 82. The culture system according to embodiment 81, wherein the orientation of the cavity is positioned to allow fluid to flow upward from the first fluid inlet and through the packed bed.

[0094] 83. The culture system according to embodiment 81 or 82, wherein the fluid outlet is located at the second end.

[0095] 84. The culture system according to any one of embodiments 81 to 83, the culture system comprising at least one port housing at least one sensor, and the control module being configured to receive data from the at least one sensor and operate the bioreactor based on the received data.

[0096] 85. The culture system according to any one of embodiments 81 to 84, the culture system comprising at least one pump for directing fluid from the cell culture medium source and / or oxygen source into the internal space.

[0097] 86. The culture system according to any one of embodiments 81 to 85, wherein the packed bed bioreactor is defined as in any one of embodiments 1 to 42.

[0098] 87. A culture system according to any one of embodiments 81 to 85, wherein the culture system is configured to perform the method according to any one of embodiments 1 to 75. Attached Figure Description

[0099] To better understand the subject matter disclosed herein and to illustrate how it can be implemented in practice, implementation methods will now be described by way of non-limiting example only, with reference to the accompanying drawings, in which: Figures 1A to 1C This is a schematic diagram of an exemplary edible filled bed (EPB) bioreactor configuration. Figure 1A It is an EPB with a uniform distribution of a single type of edible scaffold structure. Figure 1B It is a homogeneous mixture of two different edible scaffold structures. Figure 1C It is an ordered but separate distribution of two different edible scaffold structures.

[0100] Figures 2A to 2I This study demonstrates the effects of different edible scaffold materials on cell growth. Figure 2A This is a bar graph showing the fold increase in the growth of avian fibroblasts or avian adult stem cells (ASCs) on different scaffold materials at different times. The scaffold materials include fungal scaffolds, texturized soybean protein (TSP) scaffolds, texturized plant protein (TVP) scaffolds, decellularized (DC) plant stem scaffolds, DC-vegetable root scaffolds, DC-fruit scaffolds (avian fibroblasts), as well as Gelatex type 1 scaffolds and Gelatex type 2 scaffolds (ASCs). Figures 2B to 2IThese are confocal microscopy images of cells growing on scaffold materials: fungal scaffolds, tissue-modified pea protein (TPP) scaffolds; TVP scaffolds, DC plant stem scaffolds, DC-vegetable root scaffolds, DC-fruit scaffolds, Gelatex type 1 scaffolds, and Gelatex type 2 scaffolds. Cells were stained with Dapi (4',6-diamidinyl-2-phenylindole, blue) and F-actin (green); scale bar 100µm.

[0101] Figures 3A to 3C The growth of poultry ASCs on edible supports of different geometries and sizes is shown. Figure 3A This is a bar chart showing thickness measurements of supports with different geometries in different views (top and side). Hex - hexagonal support, Disc - circular support. Figure 3B This is a graph showing the glucose consumption rate (GCR) of cells grown on different scaffolds in an EPB bioreactor. Figure 3C It is a graph showing soluble proteins from cells grown on different scaffolds, the soluble proteins being determined by the quinolinic acid (BCA) assay.

[0102] Figures 4A to 4D The effects of plasma treatment on cell attachment and growth in edible scaffolds were shown. Figure 4A and Figure 4B These are confocal images of stained cellulose (CBD-GFP) on untreated and plasma-treated acellular plant scaffolds, respectively. Figure 4C These are graphs showing cell growth on plasma-treated and untreated scaffolds. Figure 4D This is a graph showing the soluble proteins in plasma-treated and untreated scaffolds.

[0103] Figures 5A to 5D The effects of different solutions on cell attachment and growth in edible scaffolds were shown. Figure 5A This is a graph showing cell growth at different times on scaffolds treated with fibronectin factor attachment solution (“attached”), spent culture medium (“used medium”), and PBS (“unattached”). Figures 5B to 5D These are confocal images of cells seeded on edible scaffolds washed with a solution. Figure 5B The scaffold was washed with a fibronectin factor attachment solution. Figure 5C It is a scaffold washed with waste culture medium from cell culture. Figure 5D The scaffold was washed with PBS; the cells were stained with Dapi (blue) and F-actin (green), scale bar 1 mm.

[0104] Figures 6A to 6HThese are confocal microscope images of different cell types grown on a scaffold in an EPB bioreactor. Figure 6A and Figure 6E Showing ASC for birds, Figure 6B and Figure 6F Showing avian fibroblasts, Figure 6C and Figure 6G Showing cow ASC, Figure 6D and Figure 6H Showing bovine fibroblasts, Figures 6A to 6D The scale is 1000µm. Figures 6E to 6H The scale bar was 100µm, and the cells were stained with Dapi (blue) and F-actin (green).

[0105] Figures 7A to 7N The cell growth distribution on and between scaffolds in the EPB bioreactor is shown. Figure 7A and Figure 7B These are confocal microscopy images of cell nuclei (stained with Dapi) and actin filaments (stained with phalloidin) in cells located on opposite sides of the scaffold. Figure 7C and Figure 7D This is an image of biomass harvested from a bioreactor, which is formed by scaffold clusters with cells cultured between these scaffolds; Figures 7E to 7N These are images showing the cell distribution at different depths of the scaffold (cells stained with Sirius red). Scaffold 1 and scaffold 2 are duplicate samples of the same type of scaffold.

[0106] Figures 8A to 8D This is a chart showing the metabolite monitoring during the growth of cells (meat) cultured in the EPB bioreactor. Figure 8A This represents the rate of glucose consumption. Figure 8B The rate of NH3 formation. Figure 8C This represents the rate of lactic acid formation. Figure 8D This represents the rate of lactate dehydrogenase (LDH).

[0107] Figures 9A to 9H This illustrates the characteristics of tissue formation and nutrient composition on an edible scaffold in an EPB bioreactor; Figure 9A Cells stained with Dapi and F-actin indicate cell growth; Figure 9B Cells stained with collagen indicate the extracellular matrix (ECM). Figure 9C Cells stained with Oil Red O indicate triglyceride droplets; Figures 9D to 9F This is a graph showing the protein content of cells grown in an EBP bioreactor, with cells grown in conical flasks or as cell pellets serving as a control. Figures 9G to 9H This displays the triglyceride (TG) content that indicates the differentiation of cells into adipocytes.

[0108] Figures 10A to 10D It is a graph showing the distribution of cells cultured on an edible scaffold; Figure 10A This is a graph showing the analysis of live cells cultured from the "top" or "bottom" region of a packed bed on an edible scaffold structure in a packed bed bioreactor, compared with two groups of (scaffolded) control cells grown in microplates using "direct" and "suspension" techniques. Figure 10B It is a graph showing the distribution of cells cultured on edible scaffolds in a large packed-bed bioreactor across different regions of the packed bed (different layers of the packed bed, different heights within the packed bed), indicated by red dots. Figure 10C It is a chart showing the number of cells in different layers of a packed bed and in a smaller bioreactor. Figure 10D It is a graph showing the number of cells at different distances from the center in a large packed bed compared to a small bioreactor.

[0109] Figures 11A to 11D This shows cells differentiating into mature muscle cells in the EPB bioreactor; Figure 11A and Figure 11B These are confocal images showing the formation of mature muscle fibers on edible scaffolds, indicated by nuclear staining (DAPI - blue) and myosin heavy chain staining (MyHC - red) at different magnifications. Figure 11C This is a Western blot image showing the presence of MyHC (band with a molecular weight of 240 kDa). Figure 11D It displays the information from... Figure 11C A graph showing the quantitative analysis of protein blots using MyHC.

[0110] Figures 12A to 12C It is the following image: Figure 12A A packed bed bioreactor is shown. Figure 12B This shows cells grown in a packed bed bioreactor. Figure 12C The image shows cultured tissue (cultured meat) being cooked.

[0111] Figure 13 It is a bar chart showing the average score of chicken odor intensity ranking in the olfactory test. Detailed Implementation

[0112] The subject matter disclosed in this invention is based on the discovery that cells and tissues can grow and differentiate effectively on edible scaffolds within a packed-bed bioreactor to provide edible tissue blocks that can form alternative food products, particularly including alternative meat products or alternative fish and seafood products.

[0113] As shown in this paper, the edible scaffold is designed to support cell growth both across its entire surface (along the periphery) and within its porous structure (in the scaffold's pores / voids / cavities), thereby creating a tightly simulated three-dimensional environment of natural tissue. Specifically, Figure 7A and Figure 7B The growth of cells on two opposite sides of an exemplary scaffold is shown. Figure 7C and Figure 7D The growth of cells between the scaffolds was shown. Figures 7E to 7N Cell growth at different depths within the scaffold is shown. These results demonstrate the unique characteristics of the scaffold, allowing cells to be uniformly distributed on and within the scaffold pores. This indicates that the scaffold is capable not only of supporting cell attachment, cell growth and proliferation, and cell differentiation on its surface, but also of supporting these processes within the pores and voids of the scaffold structure, forming a continuous three-dimensional (3D) structure that allows for the formation of interconnected tissues.

[0114] As described herein, multiple edible scaffolds are packed within a bioreactor in a unique close proximity configuration (arrangement) such that at least a portion of the surface of one scaffold is in contact with at least a portion of the surface of an adjacent scaffold, substantially eliminating spaces / gaps between the scaffolds. This close proximity arrangement allows for uniform cell distribution and nutrient delivery.

[0115] As described herein, the packed-bed bioreactor configuration allows both liquids and gases to flow around the scaffold structure, thereby immersing (enveloping) the scaffold and the cells seeded on it in nutrients and gases. This design ensures close proximity of nutrients and gases to the cells, allowing for efficient delivery of these essential components and overcoming diffusion limitations. By eliminating diffusion limitations, the bioreactor configuration ensures that all cells always have access to fresh nutrients and oxygen, while metabolic waste is effectively removed.

[0116] It is believed that, despite the scaffold being tightly packed (with virtually no voids / spaces), the bioreactor still achieves uniform flow throughout the system, ensuring a uniform and continuous distribution of nutrients and gases, as well as the removal of cellular byproducts, thus generally supporting cell growth and differentiation. As shown in Figure 8, the rates of glucose consumption, ammonia formation, lactate formation, and lactate dehydrogenase formation increased over time, indicating cell growth (proliferation) on the edible scaffold. Furthermore, as shown in Figure 9, increased collagen production was observed, indicating tissue formation. Additionally, as these figures illustrate, cells differentiated into adipocytes, evidenced by the presence of triglycerides. Moreover, as shown in Figure 11, cells were able to differentiate into myocytes, forming muscle tissue.

[0117] Interestingly, cells grown on edible scaffolds within a packed bed exhibited higher protein and triglyceride concentrations compared to cells grown in suspension on edible scaffolds (in conical flasks).

[0118] As shown in Figure 10, cell growth is not affected by the position of the scaffold within the bioreactor, indicating that the bioreactor supports uniform cell growth.

[0119] Unbound by theory, it is believed that scaffolds support the ability of cells to grow and differentiate on and within scaffolds. Together with the close proximity configuration between multiple scaffolds within a bioreactor, this promotes interactions between cells growing on adjacent scaffolds, including cell-cell signaling, cell movement, nutrient transport, or cell-cell adhesion, forming a three-dimensional network of interconnected cells, thereby leading to tissue formation.

[0120] Therefore, according to a first aspect of the present disclosure, a packed bed bioreactor is provided, the packed bed bioreactor including a cavity 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; The internal space is filled with multiple porous three-dimensional support structures; Several of the porous three-dimensional scaffold structures contain edible materials; The three-dimensional scaffold structures are filled within the cavity, such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure; and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for a substantially uniform flow of fluid from the first fluid inlet to the fluid outlet.

[0121] In the following disclosures, when references are made to a packed-bed bioreactor, it should be understood that this refers to the method, edible scaffold structure, edible cultured cell mass, food product, and culture system. Therefore, whenever features relating to a packed-bed bioreactor are provided, it should be understood that the same features are defined with respect to the method, edible scaffold structure, edible cultured cell mass, food product, and culture system, with necessary modifications.

[0122] As used herein, a packed-bed bioreactor refers to a bioreactor in which cells are grown on a solid support. The solid support is, or includes, the scaffold described herein.

[0123] The multiple porous three-dimensional scaffold structures used in this article encompass multiple scaffolds of the same or different types, as further described below.

[0124] As used herein, the term scaffold refers to a three-dimensional structure comprising materials suitable for cell adhesion and further cell growth and / or differentiation.

[0125] The scaffolds disclosed herein are characterized by their mechanical properties (e.g., stiffness, elasticity) that support cell attachment, proliferation, and differentiation into tissues such as flesh or fish. It is believed that these unique mechanical properties, along with the scaffold's unique distribution within a bioreactor, closely resemble the natural extracellular matrix environment.

[0126] Therefore, according to some implementation schemes, the stent is characterized by an elastic modulus between about 3 kPa and about 600 kPa, sometimes between about 3 kPa and about 100 kPa, and sometimes between about 100 kPa and about 600 kPa.

[0127] The term elastic modulus, also known as Young's modulus, is a measure of the hardness or stiffness of a material (such as a support structure). It 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 by which a material returns to its original shape after stress removal.

[0128] The modulus of elasticity can be experimentally measured by 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).

[0129] According to some implementation schemes, the stent is characterized by a stiffness between approximately 3 kPa and approximately 15 kPa.

[0130] As used in this article, stiffness is a measure of how well a material (e.g., a support) resists deformation under applied load and indicates how much force is required to achieve a certain amount of deformation.

[0131] Stiffness can be experimentally measured using various methods known in the art.

[0132] Unbound by theory, it is believed that the unique arrangement of the scaffolds, which are closely spaced together, can reduce scaffold stiffness to better accommodate cell growth and tissue formation.

[0133] As described herein, the scaffold structure incorporates edible materials. In some embodiments, the scaffold structure is made entirely of edible materials. According to such embodiments, the entire scaffold is edible.

[0134] This application is not limited to specific edible materials, but can be applied to a variety of edible materials, such as edible materials suitable for human or animal consumption.

[0135] like Figures 2A to 2I As shown, scaffold materials are not limited to a specific type, as a variety of materials, including scaffold materials derived from fungi, vegetables, and fruits, as well as processed materials including TSP and TVP, have been shown to support cell growth. These experiments have confirmed that a wide range of materials, both natural and synthetic, can effectively support the porous structures necessary for three-dimensional cell growth and cell interactions.

[0136] In some examples, the edible materials are not derived from animals.

[0137] In some examples, edible materials include plant materials, algae materials, fungal materials, or any combination thereof.

[0138] In some examples, the edible material is or contains plant material.

[0139] As used in this article, plant material means any substance derived from a plant, including any part thereof, or of plant origin, and encompasses one or more of seeds, leaves, stems or roots.

[0140] According to some examples, plant material can be decellularized (DC) plant tissue. Decellularized plants can be obtained by any method known in the art that removes native plant tissue and preserves the decellularized scaffold.

[0141] In some examples, the plant material is derived from legumes, grains, vegetables, fruits, nuts, or seeds.

[0142] In some implementations, the legumes are chickpeas, clover, soybeans, peas, lentils, lupins, bean trees, carob, soybeans, peanuts, tamarind, or any combination thereof.

[0143] In some implementations, the grain is wheat, rice, oats, or any combination thereof.

[0144] In some implementations, the seeds are derived from rapeseed, sunflower seeds, rice seeds, or any combination thereof.

[0145] In some examples, the edible material is fruit-derived material, vegetable-derived material, or a combination of these.

[0146] In some examples, the edible material is or contains fruit-derived material. In some examples, the fruit-derived material is from apples and / or pears. 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.

[0147] In some examples, the edible material is or contains vegetable-derived material. In some examples, the vegetable-derived material comes from one or more of onions, carrots, broccoli, cucumbers, potatoes, asparagus, cauliflower, peppers, parsley, bamboo shoots, or any combination thereof. In some examples, the onion is a green onion. In some examples, the onion is a white onion. In some examples, the onion is a red onion. In some examples, the vegetable-derived material is acellular vegetable-derived material.

[0148] In some examples, the edible material is or contains 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.

[0149] In some examples, the edible material is or contains edible stems.

[0150] In some examples, the edible material is or contains edible roots.

[0151] In some examples, the edible material is or contains insoluble dietary fiber. In some examples, the insoluble dietary fiber is or contains polysaccharides. In some embodiments, the polysaccharide is one or more of cellulose and hemicellulose.

[0152] In some examples, edible materials include cellulose materials.

[0153] In some examples, the edible material is or contains plant protein.

[0154] In some examples, the plant protein is legume protein. In some examples, the plant protein is cereal protein. In some examples, the plant protein is vegetable protein. In some examples, the protein is fruit protein.

[0155] In some examples, the edible material contains at least insoluble dietary fiber derived from fruit.

[0156] In addition to plant-based materials, edible scaffolds can also contain other materials. In some examples, edible materials include animal-derived materials.

[0157] In some examples, animal-derived materials are selected from the group consisting of: decellularized tissue, collagen, gelatin, elastin, laminin, fibronectin, hyalin, and any combination thereof.

[0158] In some examples, edible materials include a combination of plant-based and animal-derived materials.

[0159] This disclosure is not limited to any specific porous scaffold structure; the porous scaffold can be of any type, size, or shape, provided that it allows cell growth and / or differentiation. As shown below, the bioreactor is compatible with a variety of scaffolds.

[0160] like Figures 3A to 3C As shown, scaffolds of different shapes (including hexagonal and disc-shaped) and different sizes (including 0.5 mm and 1 mm) exhibited similar glucose consumption rates. Figure 3B ) and protein levels ( Figure 3C This indicates that cell activity is uniform and unaffected by the shape and size of the test scaffold.

[0161] Unbound by theory, bioreactors are thought to support cell growth on different scaffolds, independent of the physical dimensions of the scaffolds (e.g., shape and size). Such physical dimensions do not affect cellular activities such as glucose consumption and protein synthesis.

[0162] In some examples, each porous three-dimensional scaffold structure in the porous three-dimensional scaffold structure contains at least one dimension whose size is in the range of about 0.2 mm to about 500 mm, sometimes in the range of about 0.2 mm to about 300 mm, sometimes in the range of about 0.2 mm to about 100 mm, sometimes in the range of about 0.2 mm to about 50 mm, sometimes in the range of about 0.2 mm to about 10 mm, sometimes in the range of about 0.2 mm to about 5 mm, and sometimes in the range of about 0.2 mm to about 1 mm.

[0163] In some examples, the dimensions of at least one dimension of the porous three-dimensional scaffold structure are in the following ranges: between about 0.2 mm and about 300 mm, sometimes between about 1 mm and about 300 mm, sometimes between about 1 mm and about 200 mm, sometimes between about 1 mm and about 100 mm, sometimes between about 1 mm and about 50 mm, sometimes between about 1 mm and about 40 mm, sometimes between about 1 mm and about 30 mm, sometimes between about 1 mm and about 20 mm, and sometimes between about 1 mm and about 10 mm.

[0164] In some examples, each porous three-dimensional scaffold structure in the porous three-dimensional scaffold structure contains at least one dimension having the following dimensions: approximately 0.1 mm, sometimes approximately 0.2 mm, sometimes approximately 0.3 mm, sometimes approximately 0.4 mm, sometimes approximately 0.5 mm, sometimes approximately 0.6 mm, sometimes approximately 0.7 mm, sometimes approximately 0.8 mm, sometimes approximately 0.9 mm, sometimes approximately 1 mm, sometimes approximately 2 mm, sometimes approximately 3 mm, sometimes approximately 4 mm, sometimes approximately 5 mm, sometimes approximately 6 mm, sometimes approximately 7 mm, sometimes approximately 8 mm, sometimes approximately 9 mm, sometimes approximately 10 mm, sometimes approximately 15 mm, sometimes approximately 20 mm, and sometimes approximately 25 mm. The dimensions are sometimes approximately 30mm, sometimes approximately 35mm, sometimes approximately 40mm, sometimes approximately 50mm, sometimes approximately 60mm, sometimes approximately 70mm, sometimes approximately 80mm, sometimes approximately 90mm, sometimes approximately 100mm, sometimes approximately 120mm, sometimes approximately 150mm, sometimes approximately 170mm, sometimes approximately 200mm, sometimes approximately 220mm, sometimes approximately 250mm, sometimes approximately 270mm, sometimes approximately 300mm, sometimes approximately 320mm, sometimes approximately 350mm, sometimes approximately 370mm, sometimes approximately 400mm, sometimes approximately 420mm, sometimes approximately 450mm, sometimes approximately 470mm, and sometimes approximately 500mm. In some examples, each porous three-dimensional scaffold structure in the porous three-dimensional scaffold structure contains at least one dimension with a size of approximately 0.5mm.

[0165] In some examples, the porous three-dimensional scaffold structure has a first dimension and a second dimension that jointly define a plane, as well as a thickness perpendicular to that plane.

[0166] In some examples, the first and second dimensions have the same size. In other examples, the first and second dimensions have different sizes.

[0167] In some examples, at least one of the first and second dimensions is greater than the thickness. In some examples, at least one of the first and second dimensions has the same dimension as the thickness. In some examples, the first and second dimensions have the same dimension as the thickness.

[0168] In some examples, the thickness ranges from about 0.1 mm to about 2 mm. In some examples, the thickness is between about 0.3 mm and about 2 mm, sometimes between about 0.5 mm and about 2 mm, sometimes between about 0.7 mm and about 2 mm, and sometimes between about 0.9 mm and about 2 mm. In some examples, the thickness ranges from about 0.1 mm to about 1.7 mm, sometimes between about 0.3 mm and about 1.5 mm, sometimes between about 0.5 mm and about 1.5 mm, and sometimes between about 0.7 mm and about 1.5 mm.

[0169] In some examples, the thickness is at least about 0.1 mm, sometimes at least about 0.2 mm, sometimes at least about 0.3 mm, sometimes at least about 0.5 mm, sometimes at least about 0.7 mm, sometimes at least about 1 mm, sometimes at least about 1.2 mm, sometimes at least about 1.5 mm, sometimes at least about 1.7 mm, and sometimes at least about 2 mm.

[0170] In some examples, the thickness is in the range of about 0.1 mm to about 1 mm, sometimes between about 0.1 mm and about 0.9 mm, sometimes between about 0.1 mm and about 0.8 mm, sometimes between about 0.2 mm and about 0.9 mm, and sometimes between about 0.2 mm and about 0.8 mm.

[0171] In some examples, the thickness is about 0.1 mm, sometimes about 0.3 mm, sometimes about 0.5 mm, sometimes about 0.7 mm, sometimes about 1 mm, sometimes about 1.2 mm, sometimes about 1.5 mm, sometimes about 1.7 mm, and sometimes about 2 mm.

[0172] The support according to this disclosure comprises a porous material characterized by pores or voids that can be interconnected or not interconnected.

[0173] In some examples, the pores of the stent are interconnected. In some examples, at least a portion of the pores of the stent are interconnected.

[0174] In some examples, the pores of the support are not interconnected.

[0175] As described herein, the porous structure of the scaffold facilitates three-dimensional cell growth, allowing cells to proliferate throughout the entire surface periphery and within the scaffold pores. This design ensures that each cell is in contact with its neighboring cells, allowing cells to grow in multiple layers, thereby promoting cell interactions and tissue formation, as further described herein.

[0176] According to this disclosure, cells and liquids (e.g., cell culture medium) can permeate into the pores of the scaffold, allowing the cells to be embedded within the pores of the scaffold.

[0177] The support can contain pores of different sizes distributed throughout its structure.

[0178] These pores are believed to play a key role in the functionality of the scaffold, allowing cell infiltration and growth, as well as the diffusion of nutrients and waste. Variations in pore size can allow for a balance between structural integrity and biocompatibility, thereby optimizing the scaffold for its intended use.

[0179] Unbound by theory, it is believed that the pores of the scaffold can accommodate the attachment and growth of cells within the pores.

[0180] In some embodiments, the porous scaffold comprises a porous material containing pores, wherein the size of these pores is such that they can accommodate at least one cell, sometimes at least two cells, and sometimes at least ten cells.

[0181] Scaffold pore size can be measured by any method known in the art, such as the method described in the following literature: (Engineering Aligned Skeletal Muscle Tissue Using Decellularized Plant-Derived Scaffolds | ACS Biomaterials Science & Engineering). In short, the scaffold was stained with Carcorflur White M2R (1:10,000) to stain the cellulose, and imaged under a laser scanning confocal microscope to obtain a 3D reconstruction of the loaded scaffold. The cross-sectional XZ profile of the scaffold surface morphology perpendicular to the alignment direction was generated by 3D confocal z-stacking of Carcorflur White-stained cellulose.

[0182] Pore ​​size can be measured by any method known in the art. For example, pore size can be determined by measuring the diameter of a representative pore sample and determining the mean (average).

[0183] Pore ​​size can be measured by any method known in the art. For example, pore size can be determined by measuring the diameter of a representative pore sample and determining the mean (average).

[0184] When referring to pore size, it should be understood as referring to the average pore size of the support structure.

[0185] In some examples, the porous support comprises a porous material having pores with a diameter of at least about 2 µm, at least about 10 µm, or at least about 15 µm. In some examples, the porous support comprises a porous material having a diameter of at least about 20 µm, sometimes at least about 30 µm, sometimes at least about 40 µm, sometimes at least about 50 µm, sometimes at least about 60 µm, sometimes at least about 70 µm, sometimes at least about 80 µm, sometimes at least about 90 µm, sometimes at least about 100 µm, sometimes at least about 110 µm, sometimes at least about 120 µm, sometimes at least about 130 µm, sometimes at least about 140 µm, sometimes at least about 150 µm, sometimes at least about 160 µm, and sometimes... Porosity of at least about 170 µm, sometimes at least about 180 µm, sometimes at least about 190 µm, sometimes at least about 200 µm, sometimes at least about 210 µm, sometimes at least about 220 µm, sometimes at least about 230 µm, sometimes at least about 240 µm, sometimes at least about 250 µm, sometimes at least about 260 µm, sometimes at least about 270 µm, sometimes at least about 280 µm, sometimes at least about 290 µm, sometimes at least about 300 µm, sometimes at least about 350 µm, sometimes at least about 370 µm, sometimes at least about 400 µm.

[0186] In some examples, the porous support comprises a porous material containing pores with a diameter between about 2 µm and about 2 mm. In some examples, the porous support comprises a porous material containing pores with a diameter between about 10 µm and about 1 mm, sometimes between about 20 µm and about 800 µm, sometimes between about 20 µm and about 700 µm, sometimes between about 20 µm and about 600 µm, sometimes between about 20 µm and about 500 µm, sometimes between about 20 µm and about 400 µm, sometimes between about 20 µm and about 400 µm, sometimes between about 20 µm and about 300 µm, and sometimes between about 20 µm and about 300 µm.

[0187] In some examples, the porous support comprises a porous material containing pores with diameters of about 2 µm, sometimes about 10 µm, and sometimes about 15 µm. In other examples, the porous support comprises a porous material containing pores with diameters of about 20 µm, sometimes about 30 µm, sometimes about 40 µm, sometimes about 50 µm, sometimes about 60 µm, sometimes about 70 µm, sometimes about 80 µm, sometimes about 90 µm, sometimes about 100 µm, sometimes about 110 µm, sometimes about 120 µm, sometimes about 130 µm, sometimes about 140 µm, sometimes about 150 µm, sometimes about 160 µm, and sometimes... Pores approximately 170µm, sometimes 180µm, sometimes 190µm, sometimes 200µm, sometimes 210µm, sometimes 220µm, sometimes 230µm, sometimes 240µm, sometimes 250µm, sometimes 260µm, sometimes 270µm, sometimes 280µm, sometimes 290µm, sometimes 300µm, sometimes 350µm, sometimes 370µm, and sometimes 400µm.

[0188] Understandably, the porosity of the stent (i.e., the number of pores / voids in the stent structure) is important, as it can affect the flow of nutrients, gases, and waste, thus impacting the stent's function.

[0189] Porosity can be measured and calculated using various methods known in the art, including water retention capacity methods, dry porosity measurement methods, and combinations thereof.

[0190] For example, the porosity of the support can be determined using the following formula: support porosity

[0191] In some examples, the porosity of the support is at least about 40%, sometimes at least about 50%, sometimes at least about 55%, sometimes at least about 60%, sometimes at least about 65%, and sometimes at least about 70%.

[0192] In some examples, the porosity of the support is between 50% and 99%, sometimes between 50% and 95%, sometimes between 50% and 90%, sometimes between 50% and 85%, sometimes between 50% and 80%, sometimes between 50% and 75%, and sometimes between 50% and 70%.

[0193] In some examples, the stent porosity is about 50%, sometimes about 55%, sometimes about 60%, sometimes about 63%, sometimes about 65%, and sometimes about 70%. In some examples, the stent porosity is about 63%.

[0194] Furthermore, the water retention capacity of the stent can be determined using the bulk density method or the water saturation method. In some examples, the water retention capacity is calculated as follows: water holding capacity = ( Support wet weight - dry weight of support )*100 / ( Support wet weight ) .

[0195] In some examples, multiple porous three-dimensional scaffold structures have a water retention capacity (percentage of void volume in the total volume of the three-dimensional structure) in the range of approximately 50% to approximately 99%, calculated as: (wet weight of scaffold - dry weight of scaffold) * 100 / (wet weight of scaffold).

[0196] In some examples, the water retention capacity of the stent ranges from about 55% to about 99%, sometimes from about 60% to about 99%, sometimes from about 65% to about 99%, sometimes from about 70% to about 99%, sometimes from about 75% to about 99%, sometimes from about 80% to about 99%, and sometimes from about 80% to about 98%.

[0197] In some examples, the water retention capacity is about 50%, sometimes about 55%, sometimes about 60%, sometimes about 65%, sometimes about 70%, sometimes about 75%, sometimes about 80%, sometimes about 85%, sometimes about 90%, sometimes about 95%, sometimes about 96%, and sometimes about 97%.

[0198] In some examples, the water retention capacity of the stent is about 75%, sometimes about 80%, sometimes about 85%, sometimes about 90%, sometimes about 95%, sometimes about 96%, and sometimes about 97%.

[0199] The dry density of the filler used provides a measure of how tightly the stents are packed together in a dry state, and can be defined as the mass of the dry stent divided by the volume of the stent under conditions of tight filling or compaction (without the addition of water or any other fluid).

[0200] In some examples, multiple porous three-dimensional scaffold structures are filled with a dry density ranging from about 1 mg / ml to about 1000 mg / ml, sometimes from about 1 mg / ml to about 750 mg / ml, sometimes from about 1 mg / ml to about 500 mg / ml, sometimes from about 1 mg / ml to about 250 mg / ml, sometimes from about 1 mg / ml to about 100 mg / ml, sometimes from about 1 mg / ml to about 75 mg / ml, sometimes from about 1 mg / ml to about 50 mg / ml, sometimes from about 1 mg / ml to about 40 mg / ml, sometimes from about 2 mg / ml to about 40 mg / ml, sometimes from about 3 mg / ml to about 30 mg / ml, sometimes from about 4 mg / ml to about 25 mg / ml, and sometimes from about 5 mg / ml to about 20 mg / ml.

[0201] As used in this article, the wet packing density provides a measure of how tightly the stents are packed together when wetted or immersed in a liquid.

[0202] In some examples, multiple porous three-dimensional scaffold structures are filled with a wet density ranging from about 0.1 g / ml to about 1.0 g / ml, sometimes from about 0.1 g / ml to about 0.75 g / ml, and sometimes from about 0.1 g / ml to about 0.5 g / ml.

[0203] As noted in this article, scaffolds are not limited to a specific shape and can be any shape that allows multiple scaffolds to be filled in a bioreactor. In some examples, three-dimensional scaffold structures have shapes selected from the group consisting of sheet-like, fibrous, bead-like, sheet-like, disk-like, spherical, cylindrical, toroidal, polygonal, and star-shaped forms.

[0204] In some examples, the three-dimensional scaffold structure has a square shape. In some examples, the three-dimensional scaffold structure has a circular shape. In some examples, the three-dimensional scaffold structure has a hexagonal shape. In some examples, the three-dimensional scaffold structure does not have a fibrous 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.

[0205] The scaffold according to this disclosure is configured to allow a cell density of 20k to 100k cells per milligram of wet scaffold.

[0206] According to this disclosure, the edible scaffold is characterized by its ability to provide texture and color to the resulting alternative food.

[0207] like Figure 13As shown, cultured meat products prepared in EBP (containing 6.8% cells) exhibit a higher sensory effect compared to mixed products composed of the same percentage of cytoplasm. Furthermore, cultured meat products prepared in EBP (containing 6.8% cells) are characterized by having the same sensory effect as mixed products composed of 50% cytoplasm. These results indicate that cell and tissue growth in EBP is advantageous because it provides desirable sensory properties at low cell density. This suggests that the increased cell production capacity and tissue yield in EBP are associated with the release of volatile compounds that mimic the sensory characteristics of conventional food products.

[0208] As described herein, multiple scaffolds within a packed-bed bioreactor are adapted to support cell attachment, cell growth and proliferation, and / or differentiation. In some examples, the packed-bed bioreactor is used in conjunction with a culture medium source for culturing cells within the cavity.

[0209] To improve the effectiveness of the scaffold, the scaffold can be pretreated, that is, treated before cell seeding.

[0210] These treatments may involve altering the surface and / or porosity properties of the scaffold, particularly its mechanical strength and surface characteristics, in order to enhance its performance and suitability for cell growth and / or cell differentiation.

[0211] In some examples, the porous three-dimensional scaffold structure is pre-adjusted to facilitate cell adhesion to the scaffold structure.

[0212] The treatment of the scaffold before cell seeding is referred to as preconditioning in this paper.

[0213] In some examples, the physical and / or chemical alterations of the surfaces, including the porous three-dimensional scaffold structure, are pre-adjusted.

[0214] In some examples, physical modifications to the surfaces of the porous three-dimensional scaffold structure are pre-adjusted. As used herein, the term "physical modification to the surface of a porous three-dimensional scaffold structure" refers to any alteration or modification to the physical properties of at least one surface of the scaffold while maintaining its porous and three-dimensional properties. Physical properties may include one or more of the texture or morphology of the scaffold surface, and alterations to one or more physical properties may be achieved, for example, by surface roughening, smoothing, patterning, or etching.

[0215] In some examples, the chemical modifications to the surface of the porous three-dimensional scaffold structure are pre-adjusted. As used herein, the term "chemical modification of the surface of a porous three-dimensional scaffold structure" refers to any alteration or modification that occurs in the surface chemistry and composition of the porous three-dimensional scaffold structure, such as by introducing new chemical functions or altering the existing functions of the scaffold, while maintaining its porous and three-dimensional properties. Chemical modifications may involve attaching chemicals, molecules, or polymers to the scaffold surface via covalent or non-covalent bonds.

[0216] In some examples, the pre-adjustment includes any of the following: plasma treatment or electrostatic charging of the surface of the porous three-dimensional scaffold structure; increasing the surface energy of the porous three-dimensional scaffold structure; removing lignin from the surface of the porous three-dimensional scaffold structure; or exposing cellulose residues on the surface of the porous three-dimensional scaffold structure.

[0217] In some examples, pre-treatment includes plasma processing.

[0218] In some examples, the porous three-dimensional scaffold structure is pre-adjusted to expose cellulose residues on the surface of the porous three-dimensional scaffold structure.

[0219] In some examples, the porous three-dimensional scaffold structure is pre-adjusted to remove lignin from the surface of the porous three-dimensional scaffold structure.

[0220] In some examples, vacuum plasma treatment or corona treatment is used to pre-adjust the porous three-dimensional scaffold structure.

[0221] In some examples, vacuum plasma treatment is used to pre-adjust the porous three-dimensional scaffold structure.

[0222] In some examples, the porous three-dimensional scaffold structure is a plasma-treated scaffold.

[0223] As shown in Figure 4, cell growth and protein content both increased in the plasma-treated scaffold.

[0224] In some examples, the porous three-dimensional scaffold structure is pre-adjusted to increase the positive charge on the surface of the porous three-dimensional scaffold structure.

[0225] Regardless of the preconditioning method used, it should be noted that preconditioning is determined by the level of cell adhesion on the porous three-dimensional scaffold structure. Preconditioning can be determined by assessing the percentage of positive surface charge and exposed cellulose binding sites. Cellulose binding sites can be measured by any method known in the art, such as fluorescence-based assays, where fluorescently labeled cellulose-binding molecules can be used to measure cellulose binding sites.

[0226] In some examples, the porous three-dimensional scaffold structure is washed with an adhering solution.

[0227] In some examples, the porous three-dimensional scaffold structure is washed after pre-adjustment.

[0228] In some examples, the porous three-dimensional scaffold structure is washed with an adhering solution.

[0229] As used in this article, the attachment solution refers to a cell culture medium that enhances / promotes cell attachment / adhesion to the surface of a porous scaffold. As described herein, the attachment solution is used to maximize cell adhesion to the scaffold surface.

[0230] The attachment solution contains one or more biomolecules or derivatives thereof that can interact with cell surface receptors, and can therefore be considered as an attachment factor for enhancing cell interaction.

[0231] 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, fibronectin attachment solution, elastin attachment solution, poly-L-lysine attachment solution, gelatin attachment solution or used culture medium attachment solution, derivatives or combinations thereof.

[0232] In some examples, no attachment material was used, and the scaffold was washed with PBS or fresh culture medium.

[0233] It should be noted that the use of one or more of the attachment solutions can help cells attach and spread on the scaffold surface.

[0234] As shown in Figure 5, washing the scaffold with attachment solution or used culture medium before cell seeding effectively enhances the scaffold's ability to support cell attachment and proliferation. In some cases, washing the scaffold with PBS is sufficient to support cell attachment and proliferation.

[0235] In some examples, the porous three-dimensional scaffold structure is a freeze-dried structure.

[0236] In some examples, the porous three-dimensional scaffold structure is sterilized. The scaffold can be sterilized at any time, such as before filling into the bioreactor or after introduction into the bioreactor.

[0237] In some examples, the porous three-dimensional scaffold structure is sterilized within the bioreactor.

[0238] Sterilization can be performed by any method known in the art. In some examples, the porous three-dimensional scaffold structure is sterilized by any sterilization method selected from steam or autoclaving heating, ultraviolet irradiation, radiation sterilization, and dry heat sterilization.

[0239] In some examples, bioreactors containing scaffolds are sterilized using a dry autoclave.

[0240] As detailed above, multiple scaffolds are placed within the bioreactor to allow for any of the processes involved in cell attachment, growth, differentiation, and tissue formation.

[0241] As described in this article, bioreactors are highly adaptable because they can accommodate a single scaffold type or multiple scaffold types arranged 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 homogeneous or heterogeneous (non-homogeneous, ordered) environments to optimally support the growth of various tissues.

[0242] For example, as described below, scaffolds designed for muscle tissue growth can be arranged to provide the necessary stiffness and structure, while scaffolds designed for adipose tissue formation can be positioned to ensure flexibility and nutrient availability.

[0243] Furthermore, because these different scaffold types can be distributed in a uniform or non-uniform (ordered) manner, different cultured food products can be produced.

[0244] This capability enables bioreactors to produce a variety of food products, including multi-textured food products.

[0245] Scaffolds, whether identical or different, can be assembled in several configurations within a bioreactor. (Reference) Figure 1A As a schematic diagram, a bioreactor with a uniform distribution of a single type of scaffold structure is shown. Figure 1B and Figure 1C Two different types of bioreactors with varying scaffold structures and distributions are shown, in which... Figure 1B This shows a basically uniform distribution / mixture of two different scaffolds in a bioreactor, while Figure 1C The regions within the bioreactor are shown, with each scaffold structure occupying a distinct area (unmixed scaffold structure population).

[0246] Therefore, multiple stent structures can include a single type of stent structure or at least two different types of stent structures.

[0247] In some examples, multiple scaffold structures contain a single type of scaffold. In some examples, multiple scaffold structures containing a single type of scaffold are arranged in a uniform distribution within the bioreactor. The uniform distribution of the single type of scaffold... Figure 1A As shown in the diagram. In some examples, multiple scaffold structures include a single-type scaffold that can be used to generate a single type of cell. The choice of a single-type scaffold depends on the desired cell and tissue type.

[0248] In some examples, the bioreactor comprises multiple scaffold structures characterized by an elastic modulus of at most about 100 kPa, sometimes between about 3 kPa and about 100 kPa. In some examples, the multiple scaffold structures characterized by an elastic modulus of at most about 100 kPa, sometimes between about 3 kPa and about 100 kPa, are suitable for the growth of adipose tissue, liver tissue, or connective tissue.

[0249] In some examples, the bioreactor comprises multiple scaffold structures characterized by an elastic modulus of at least about 100 kPa, and sometimes between about 100 kPa and about 600 kPa. In some examples, the multiple scaffold structures characterized by an elastic modulus of at least about 100 kPa, and sometimes between about 100 kPa and about 600 kPa, are suitable for the growth of muscle, cartilage, or bone tissue.

[0250] It should be noted that using different types of scaffolds may help support the growth and / or differentiation of different cell types. Therefore, a variety of scaffolds with different properties, such as pore size, porosity, surface chemistry, and mechanical strength, can be used to provide an optimal environment for the growth and / or differentiation of different cell types within a packed bed bioreactor.

[0251] When referring to different types of stents, it should be understood that the stents defined as different types differ in at least one stent characteristic, including stent material, stent size, stent shape, stent stiffness, stent elasticity, stent average pore size, stent porosity, stent color, or stent texture.

[0252] In some examples, multiple support structures contain at least two different types of supports, wherein the at least two different types differ in at least one, sometimes at least two, and sometimes at least three, of support material, support size, support shape, support stiffness, support elasticity, support average pore size, support porosity, support color, support texture, or any combination thereof.

[0253] In some examples, multiple scaffold structures contain at least two different types of scaffolds, wherein the at least two different types contain at least different edible materials.

[0254] In some examples, multiple support structures contain at least two different types of supports, wherein the at least two different types have at least different support sizes.

[0255] In some examples, multiple support structures contain at least two different types of supports, wherein the at least two different types have at least different support shapes.

[0256] In some examples, multiple support structures contain at least two different types of supports, wherein the at least two different types have at least different support stiffnesses.

[0257] In some examples, multiple support structures contain at least two different types of support, wherein the at least two different types have at least different support elasticities.

[0258] In some examples, multiple support structures contain at least two different types of supports, wherein the at least two different types have at least different average pore sizes.

[0259] In some examples, multiple scaffold structures contain at least two different types of scaffolds, wherein the at least two different types have at least different scaffold porosities.

[0260] In some examples, multiple support structures contain at least two different types of supports, wherein the at least two different types have at least different support colors.

[0261] In some examples, multiple scaffold structures contain at least two different types of scaffolds, wherein the at least two different types have at least different scaffold textures.

[0262] The arrangement of multiple scaffolds within a bioreactor can vary depending on a variety of factors, including the tissue to be formed, the bioreactor configuration, the texture of the culture product, and the color of the culture product.

[0263] In some examples, multiple support structures are arranged in a uniform distribution.

[0264] In some examples, multiple support structures contain a single type of support, wherein the support structures are arranged in a uniform distribution.

[0265] In some examples, multiple scaffold structures contain at least two different types of scaffolds, which can be arranged in a uniformly mixed distribution within the bioreactor.

[0266] As used herein, a uniform distribution (arrangement) of a single type of scaffold or at least two different types of scaffolds in a bioreactor refers to a uniform and consistent arrangement of that particular scaffold throughout a given space or medium within the bioreactor. This means that the scaffolds are uniformly distributed, ensuring that their properties and functions are equally available in all areas of the bioreactor. In the 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 volume or region.

[0267] A homogeneous mixture of two different types of scaffolds in Figure 1B As shown in the image.

[0268] In some examples, multiple scaffold structures contain at least two different types of scaffolds, which are arranged non-uniformly within the bioreactor.

[0269] As used herein, an ordered distribution (arrangement) of two or more different types of scaffolds refers to an intentionally tailored arrangement in which the various scaffold types are not uniformly dispersed throughout the bioreactor. In other words, different scaffold types are placed in specific areas to optimize the function of the bioreactor and meet specific biological and mechanical requirements.

[0270] The orderly distribution of two different types of stents Figure 1C As shown in the image.

[0271] In some examples, the arrangement of at least two different types of scaffolds depends on the resulting tissue and the resulting alternative food products.

[0272] In some examples, the bioreactor comprises multiple scaffold structures arranged in a uniform distribution, including at least two different types of scaffolds with different elasticities.

[0273] In some examples, the bioreactor comprises multiple 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 100 kPa, and at least another different type of scaffold is characterized by an elastic modulus of at least about 100 kPa.

[0274] In some examples, the bioreactor comprises multiple scaffold structures, including at least two different types of scaffolds, wherein at least one type of scaffold is characterized by an elastic modulus between about 3 kPa and about 100 kPa, and at least another different type of scaffold is characterized by an elastic modulus between about 100 kPa and about 600 kPa.

[0275] In some examples, scaffolds characterized by an elastic modulus of up to about 100 kPa may be suitable for cell growth and differentiation into soft tissue.

[0276] In some examples, scaffolds with an elastic modulus of up to about 100 kPa may be suitable for the growth and differentiation of adipose tissue, liver tissue, or connective tissue. In some examples, scaffolds with an elastic modulus between about 3 kPa and about 100 kPa may be suitable for the growth and differentiation of adipose tissue, liver tissue, or connective tissue.

[0277] In some examples, scaffolds characterized by an elastic modulus of at least about 100 kPa may be suitable for cell growth and differentiation into rigid tissues.

[0278] In some examples, scaffolds with an elastic modulus of at least about 100 kPa may be suitable for the growth and differentiation of muscle or bone cells. In some examples, scaffolds with an elastic modulus between about 100 kPa and about 600 kPa may be suitable for the growth and differentiation of muscle and bone cells.

[0279] With these scaffolds of varying elasticity evenly distributed, strong yet flexible tissue components can be uniformly cultured and formed throughout the bioreactor. This scaffold assembly supporting the growth of muscle, fat, and bone tissue can provide the structure and arrangement for muscle fibers along with fat cells and bone cells.

[0280] In some examples, the bioreactor includes multiple scaffold structures that can be arranged in a non-uniform, ordered distribution within the bioreactor, including at least two different types of scaffolds with different elasticities. In some examples, a scaffold with an elastic modulus of at most about 100 kPa is characterized in that it can accommodate the internal portion of the bioreactor, while a scaffold with an elastic modulus of at least about 100 kPa is characterized in that it can accommodate the top and bottom portions of the bioreactor.

[0281] In some examples, the bioreactor includes multiple scaffold structures that can be arranged in a uniform and / or non-uniform ordered distribution within the bioreactor, including at least two different types of scaffolds with different sizes. In some examples, scaffolds with a size of up to 6 mm are characterized in that they can accommodate the internal portion of the bioreactor, while scaffolds with a size of at least 6 mm can accommodate the top and bottom portions of the bioreactor to support the mechanical load and function of the packed bed and to allow the culture medium to flow within the void volume.

[0282] In some examples, the bioreactor comprises multiple scaffold structures, including at least two different types of scaffolds with different colors, the colors of which are determined such that each scaffold type can mimic the resulting food substitute tissue.

[0283] The packing characteristics of porous scaffold structures in packed bed bioreactors, such as packing density, may depend on the porosity of the scaffold and may affect the activity and performance of the bioreactor in terms of cell growth and differentiation. Packing density is generally defined as the ratio of the scaffold structure volume to the total volume of the bioreactor and can be measured under dry or humid conditions.

[0284] For example Figure 12A The bioreactor described herein has a first end (120) and a second end (140), as well as an internal space (160) filled with a plurality of porous three-dimensional scaffold structures (180).

[0285] In some examples, at least one fluid inlet and / or at least one fluid outlet form part of the bioreactor.

[0286] The fluid inlet is configured to deliver culture medium, with or without cells, into the bioreactor, and the fluid outlet is configured to remove waste culture medium from the bioreactor.

[0287] Packed bed reactors may include additional components. In some examples, packed bed bioreactors include a removable top plate configured to allow the introduction and / or removal of the chamber contents.

[0288] As described herein, a packed-bed bioreactor includes means for determining the condition of cells throughout their growth and / or differentiation process. Therefore, the bioreactor includes means that allow monitoring of this condition, for example, via specific sensors. In some examples, a packed-bed bioreactor includes at least one port for housing at least one sensor.

[0289] This disclosure is not limited to specific sensors; the bioreactor may contain one or more sensors configured to monitor cell status by determining at least one culture medium parameter.

[0290] In some examples, 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, lactic acid, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH), biomass weight, biomass level in the cavity, and foam.

[0291] In some examples, one or more sensors are sterilized sensors. In some examples, one or more sensors may be sterilized within the bioreactor. In some examples, one or more sensors may be sterilized before being placed inside the bioreactor.

[0292] In some examples, the sensors are sterilized individually and then aseptically placed into the bioreactor. In some examples, the pH and dissolved oxygen (DO) sensors are sterilized individually and then aseptically placed into the bioreactor. In some examples, the pH and dissolved oxygen (DO) sensors are autoclaved.

[0293] According to some examples, a bioreactor includes a mechanism configured to allow fluid to circulate within the internal space of the bioreactor. In some examples, the mechanism includes at least one component selected from the group consisting of: impeller, peristaltic pump, diaphragm pump, vibrator, tilter, and lifter (hydraulic head).

[0294] Fluid circulation can vary depending on the size of the bioreactor and the amount of scaffolds and cells deposited within it. In some examples, the fluid circulation rate ranges from approximately 0.1 to 3 times the working volume of the bioreactor's liquid culture medium per minute.

[0295] The bioreactor is configured to allow fluid to flow into the chamber via an inlet. In some examples, the orientation of the chamber is positioned to allow fluid to flow upward from the fluid inlet and through the packed bed.

[0296] As used herein, the term flow encompasses culture medium flow, which refers to a configuration of a bioreactor in which culture medium entering through an inlet comes into close contact with the scaffold and cells within the bioreactor.

[0297] The bioreactor includes 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 cell growth and / or differentiation.

[0298] In some examples, the cavity is made of a biocompatible material. In some examples, the cavity is made of a polymer. In some examples, the polymer is one or more of polypropylene, polyvinyl chloride, polycarbonate, and polystyrene.

[0299] In one example, the cavity is made of glass. In some examples, the cavity is made of stainless steel.

[0300] In some examples, the walls of the cavity include at least one transparent segment configured to allow observation of the contents of the interior space.

[0301] The scaffold is filled within the packed bed bioreactor and serves as a platform for cell seeding, cell growth and / or cell differentiation, and tissue formation.

[0302] This disclosure is not limited to a specific cell type or a specific cell source, and is applicable to a variety of cell types provided that they have the ability to attach to multiple scaffolds.

[0303] like Figures 6A to 6H As shown, several cell types are able to adhere to the scaffold and proliferate on it.

[0304] In some examples, the cells are adherent cells.

[0305] In some examples, the cells are non-human cells. In some examples, the cells are from animal (non-human) sources. In some examples, the cells are adherent (non-human) cells. In some examples, the cells are unmodified. In some examples, the cells are genetically modified.

[0306] As described in this article, based on some examples, cells are used to produce alternative cultured food products, such as alternative cultured meat or alternative cultured fish and seafood.

[0307] In some examples, the cells are derived from mammals.

[0308] In some examples, the cells are derived from non-human mammals. In other examples, the cells are non-human cells.

[0309] In some examples, the cells were derived from poultry.

[0310] In some examples, the cells are from poultry. In some examples, the cells are from poultry eggs. In some examples, the cells are poultry cells. In some examples, the cells are poultry egg cells.

[0311] In some examples, the cell is any of a chicken cell, a duck cell, or a turkey cell.

[0312] In some examples, the cells came from cattle.

[0313] In some examples, the cells are derived from cattle, pigs, sheep, rabbits, equines, canines, or any combination thereof.

[0314] In some examples, the cells are obtained from the placenta. In some examples, the cells are placental cells.

[0315] In some examples, the cells are obtained from the embryo.

[0316] In some examples, the cells are chicken embryonic fibroblasts.

[0317] In some examples, the cells are duck embryonic fibroblasts. In other examples, the cells are derived from aquatic animals.

[0318] In some examples, the cells come from one or more of salmon, tuna, shrimp, or lobster.

[0319] In some examples, the cells contain at least stem cells.

[0320] As used in this article, stem cells refer to undifferentiated cells that have the ability to develop into various specialized cell types.

[0321] In some examples, the cells are selected from the group consisting of: embryonic stem cells, induced pluripotent stem cells, pluripotent stem cells, adult stem cells, mesenchymal stem cells, myoblasts, satellite myoblasts, fibroblasts, hepatocytes, osteoblasts, chondrocytes, adipocytes, hepatocytes, osteoblasts, chondrocytes, any combination of these, and any other natural and genetically modified adherent cells.

[0322] In some examples, the cells are avian embryonic stem cells. In some examples, the cells are duck embryonic stem cells. In some examples, the cells are bovine embryonic stem cells.

[0323] In some examples, the cells are avian adipose-derived stem cells (pASCs). In some examples, the cells are avian fibroblasts.

[0324] In some examples, the cells are bovine ASC cells (bASC). In some instances, the cells are bovine fibroblasts.

[0325] As described in this article, cells are allowed to grow on scaffold structures for the purpose of cell growth and / or differentiation.

[0326] In some examples, cells can be seeded on a scaffold within a bioreactor. In other words, the scaffold is first placed in the bioreactor, then cells are added to the bioreactor to allow contact between the cells and the scaffold, and the cells are seeded onto the scaffold and into the scaffold pores as described herein.

[0327] Therefore, according to a second aspect of the subject matter disclosed in the present invention, a method is provided, the method comprising: (i) Introducing a cell culture medium containing cells into a packed bed bioreactor, the bioreactor including a cavity 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 also including at least one fluid inlet at the first end and at least one fluid outlet. The internal space is filled with multiple porous three-dimensional support structures; Several of the porous three-dimensional scaffold structures contain edible materials; The three-dimensional scaffold structures are filled within the cavity, such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure; and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet. (ii) Providing conditions that support the growth and / or differentiation of the cells on the porous three-dimensional scaffold structure; and (iii) Harvest the porous three-dimensional scaffold structure containing cells thereon.

[0328] As described herein, to increase cell attachment and thus maximize cell seeding on and within the scaffold pores, the scaffold can be treated prior to cell seeding. In such cases, the method may include a step of treating the scaffold prior to cell seeding. This treatment of the scaffold prior to cell seeding is referred to herein as preconditioning.

[0329] In some examples, the method includes pre-adjusting the support. In some examples, pre-adjustment includes plasma treatment. In some examples, pre-adjustment includes vacuum plasma treatment.

[0330] In some examples, the method involves pre-adjusting multiple scaffold structures prior to cell seeding.

[0331] In some examples, the method includes at least one pre-adjustment step of multiple scaffold structures prior to cell seeding, wherein this pre-adjustment is performed outside the bioreactor. In other words, the multiple scaffold structures undergo at least one pre-adjustment step before being introduced and filled into the bioreactor.

[0332] In some examples, the method includes at least one pre-adjustment step of multiple scaffold structures prior to cell seeding, wherein this pre-adjustment is performed inside a bioreactor. In other words, multiple scaffold structures are introduced and filled into the bioreactor, followed by at least one pre-adjustment step.

[0333] In some examples, the method includes plasma treatment of multiple scaffold structures prior to cell seeding.

[0334] In some examples, the method includes plasma treatment of multiple scaffold structures prior to cell seeding, wherein the plasma treatment is performed outside the bioreactor. In other words, multiple scaffold structures are plasma-treated before being introduced and filled into the bioreactor.

[0335] In some examples, the method includes plasma treatment of multiple scaffold structures prior to cell seeding, wherein the plasma treatment is performed inside a bioreactor. In other words, multiple scaffold structures are introduced and filled into the bioreactor, and then subjected to plasma treatment.

[0336] In some examples, the method includes performing at least one washing step on multiple scaffold structures prior to cell seeding.

[0337] In some examples, the method includes performing at least one washing step on multiple support structures after pre-adjustment.

[0338] In some examples, the method includes at least one pre-adjustment step and at least one washing step for multiple support structures.

[0339] In some examples, the method includes performing at least one washing step on multiple support structures after plasma treatment.

[0340] In some examples, the method includes plasma treatment of multiple support structures and at least one washing step.

[0341] At least one washing step can be performed using any of the adhesion solutions described herein.

[0342] In some examples, the method includes sterilizing the stent. Sterilization can be performed by any of the methods described herein.

[0343] As noted herein, the method involves introducing a cell culture medium containing cells into a packed bed bioreactor.

[0344] As used herein, cell culture medium is also referred to as cell culture medium, meaning a nutrient-rich solution used in accordance with this disclosure to support one or more processes of cell growth, proliferation and differentiation.

[0345] The method disclosed herein includes introducing a cell culture medium into a bioreactor such that the cell culture medium contains cells.

[0346] As described herein, this disclosure applies to a variety of cell types, and therefore the specific composition of the cell culture medium can be tailored to support such cells. Thus, a suitable cell culture medium may depend on the cell type and typically contains growth factors, antibiotics, nutrients, or any combination thereof.

[0347] In some examples, the cell culture medium contains a single cell type.

[0348] In some examples, the cell culture medium contains more than one cell type.

[0349] In some examples, the cell culture medium contains two or more types of non-human cells. It should be noted that the choice of one or more cell types used may depend on the cell's differentiation potential and the alternative food to be produced, such as meat and the specific cut of meat to be produced.

[0350] It should be noted that in some cases where more than one cell type is used, different types of scaffolds can be placed in the bioreactor to support the growth and function of these different cells.

[0351] In some examples, the cell culture medium contains at least stem cells.

[0352] In some examples, the cell culture medium contains 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 myoblasts, fibroblasts, hepatocytes, osteoblasts, chondrocytes, adipocytes, hepatocytes, osteoblasts, chondrocytes, any combination of these, and any other natural and genetically modified adherent cells.

[0353] In some examples, the cell culture medium contains one or more of avian ASC cells, avian fibroblasts, bovine ASC cells, bovine fibroblasts, or any combination thereof. In some examples, the cell culture medium contains one or more of stromal cells, endothelial cells, adipocytes, hematopoietic cells, myocytes, hepatocytes, chondrocytes, osteocytes, and fibroblasts.

[0354] The number of cells in a cell culture medium depends on a variety of factors, including the number of scaffolds, their spatial orientation, and their properties (such as porosity). It is understood that the higher the porosity of the scaffold, the more cells can be seeded on it, thus increasing the number of cells in the cell culture medium introduced into the bioreactor.

[0355] According to some examples, the method includes introducing cell culture medium via at least one fluid inlet of the bioreactor.

[0356] In some examples, the method includes introducing cell culture medium via at least one fluid inlet under conditions that support cell inoculation and growth.

[0357] After the cell culture medium containing cells has been introduced into the bioreactor, the cells are allowed to attach to the scaffold (i.e., cell seeding) and grow under conditions that allow for such growth.

[0358] The conditions that allow or support cell growth on and within porous three-dimensional scaffold structures encompass both conditions supporting cell seeding and conditions supporting cell growth.

[0359] As used in this article, cell seeding refers to the attachment / adhesion of cells to the scaffold surface and / or the pores of the scaffold surface, sometimes referred to as the seeding period in this article.

[0360] The number of cells seeded depends, for example, on the scaffold density and is determined such that the cells at least partially cover at least one surface of the plurality of scaffolds and / or at least partially cover at least a portion of the voids / pores of the plurality of scaffolds.

[0361] In some examples, inoculation may include one or more cell inoculation steps. In some examples, the method includes a single inoculation step. In some examples, the method includes sequential inoculation steps. In some examples where the method includes sequential cell inoculation steps, the cells in each step may be the same or different.

[0362] In some examples, conditions supporting cell seeding may include flow rate, cell concentration, and culture medium volume.

[0363] In some examples, the method includes introducing cell culture medium at a flow rate between about 1 cm / s and 5 cm / s. In some examples, the method includes introducing cell culture medium at a flow rate of about 1 cm / s, sometimes about 2 cm / s, sometimes about 3 cm / s, sometimes about 4 cm / s, and sometimes about 5 cm / s. Suitablely, this flow rate allows cells to attach to the scaffold structure (both on and within the pores), and is therefore suitable for cell seeding.

[0364] During cell seeding, agitation or vibration may be applied to maximize seeding uniformity. The rotation speed and duration of agitation or vibration may vary depending on the number of cells and scaffolds introduced into the bioreactor.

[0365] In some examples, the method includes applying vibration with an amplitude between about 0.1 cm and about 10 cm. In some examples, the method includes applying vibration with a frequency between about 0.1 Hz and about 4 Hz. In some examples, the method includes applying vibration continuously. In some examples, the method includes applying vibration intermittently.

[0366] In some examples, the method includes applying agitation during inoculation. In some examples, the method includes agitation at one rotational speed. In some examples, the method includes agitation at two rotational speeds.

[0367] In some examples, the method includes stirring at a first rotational speed ranging from about 10 rpm to about 50 rpm. In some examples, the method includes stirring at a first rotational speed of about 10 rpm, sometimes about 20 rpm, sometimes about 30 rpm, sometimes about 40 rpm, and sometimes about 50 rpm.

[0368] In some examples, the method includes stirring at a second speed between approximately 100 rpm and approximately 200 rpm. In some examples, the method includes stirring at a second speed of approximately 100 rpm, sometimes approximately 130 rpm, sometimes approximately 150 rpm, sometimes approximately 170 rpm, and sometimes approximately 200 rpm.

[0369] In some examples, the method includes stirring at alternating speeds, including a first speed ranging from about 10 rpm to about 50 rpm, and a second speed between about 100 rpm and about 200 rpm.

[0370] As this article points out, the duration of the stirring step can vary depending on various factors, especially the rotation speed.

[0371] In some examples, the method includes stirring at a first rotational speed for a period of time between about 10 minutes and about 20 minutes. In some examples, the method includes stirring at a first rotational speed for a period of time of about 10 minutes, sometimes about 15 minutes, and sometimes about 20 minutes.

[0372] In some examples, the method includes a period of stirring at a second rotational speed for less than one minute. In some examples, the method includes a period of stirring at a second rotational speed for approximately 10 seconds, sometimes approximately 20 seconds, sometimes approximately 30 seconds, sometimes approximately 40 seconds, and sometimes approximately 50 seconds.

[0373] In some examples, the method includes: stirring at a first rotational speed for a period of time between 10 and 20 minutes, and stirring at a second rotational speed for a period of time less than 1 minute.

[0374] As described in this article, the cells to be inoculated have the ability to grow, proliferate and differentiate under appropriate conditions to generate a variety of cells and tissues.

[0375] As used herein, cell growth refers to the stage after cells have been seeded onto at least one surface of a plurality of scaffolds and / or into the pores of the plurality of scaffolds.

[0376] During the growth phase, the number and / or size of cells increase due to cell proliferation.

[0377] The period during which cells are allowed to proliferate in a bioreactor is referred to as the growth period in this paper.

[0378] According to this disclosure, the method provides conditions that support cell growth.

[0379] It is understood that conditions during the growth phase are optimized to suit the cells growing in the bioreactor and may vary depending on, for example, the type of cells introduced into the bioreactor, including, in particular, nutrients, temperature, pH, osmotic pressure, and gas exchange. In some examples, the conditions supporting cell growth on a porous three-dimensional scaffold structure include at least one condition selected from the group consisting of: temperature, pH, dissolved oxygen (DO), and agitation.

[0380] In some examples, the cell culture medium used during the growth phase is the same as that used during the inoculation phase. In other examples, the cell culture medium used during the growth phase is different from that used during the inoculation phase.

[0381] In some examples, the cell culture medium during the growth phase is suitable for the growth of non-human animal cells.

[0382] In some examples, the cell culture medium during the growth phase contains one or more of serum (e.g., fetal bovine serum (FBS)), nutrients, amino acids, antibiotics, or any combination thereof.

[0383] In some examples, the cell culture medium during the growth phase contains DMEM / F-12, FBS, GlutaMAX, and gentamicin.

[0384] In some examples, the method includes monitoring at least one parameter selected from the group consisting of: pH, dissolved oxygen (DO), temperature capacitance, metabolites, biomass weight, biomass level in the cavity, and foam during cell growth.

[0385] In some examples, the metabolites include at least one metabolite selected from the group consisting of glucose, lactate, glutamine, glutamate, NH3, and lactate dehydrogenase (LDH).

[0386] In some examples, the metabolites include at least one metabolite selected from the group consisting of glucose, lactate, NH3, and lactate dehydrogenase (LDH).

[0387] In some examples, the method includes monitoring the rate of glucose consumption.

[0388] In some examples, the method includes monitoring the rate of lactate formation.

[0389] In some examples, the method includes monitoring the ammonia formation rate.

[0390] In some examples, the method includes monitoring the LDH formation rate.

[0391] In some examples, the method includes applying a temperature ranging from about 20°C to about 45°C. In some examples, the method includes applying a temperature ranging from about 25°C to about 40°C, and sometimes from about 30°C to about 40°C. In some examples, the method includes applying a temperature of about 25°C, sometimes about 30°C, sometimes about 35°C, sometimes about 37°C, and sometimes about 39°C.

[0392] In some examples, the method involves maintaining the pH in a range between approximately 6 and approximately 8.

[0393] In some examples, the method includes maintaining the DO level in a range between about 10% and about 100%, sometimes between about 20% and about 90%, sometimes between about 20% and about 80%, and sometimes between about 30% and about 70%.

[0394] In some examples, the method includes having a value between approximately 0.1 dyn / cm 2 With approximately 10 dyn / cm 2 Between, and sometimes between, approximately 0.1 dyn / cm 2 With approximately 5 dyn / cm2 Between, and sometimes between, approximately 0.1 dyn / cm 2 With approximately 1 dyn / cm 2 The shear stress between the stirring or vibration.

[0395] In some examples, the method includes vibration with an amplitude between about 0.1 cm and about 10 cm during cell growth. In some examples, the method includes vibration with a frequency between about 0.1 Hz and about 4 Hz. In some examples, the vibration is applied continuously. In some examples, the method includes applying the vibration intermittently.

[0396] In some examples, the method includes stirring at a speed ranging from about 100 rpm to about 200 rpm during cell growth. In some examples, the method includes stirring at a speed of about 100 rpm, sometimes about 150 rpm, and sometimes about 200 rpm.

[0397] In some examples, the method includes applying a flow rate between about 5 cm / s and 30 cm / s during cell growth. In some examples, the method includes applying a flow rate of about 5 cm / s during cell growth, sometimes about 10 cm / s, sometimes about 15 cm / s, sometimes about 20 cm / s, sometimes about 25 cm / s, and sometimes about 30 cm / s.

[0398] As shown in the following example, an increased rate of glucose consumption, as well as an increased rate of lactic acid, ammonia, and LDH formation, was observed during the growth phase. Furthermore, an increase in protein content was observed during this growth phase.

[0399] It is understandable that when cell growth is mentioned, it should be understood as allowing cell proliferation, resulting in an increase in the percentage of cell coverage.

[0400] The cell coverage of a scaffold can be expressed as the cell density of each scaffold.

[0401] In some examples, the cell density per scaffold is at least about 0.1 million cells / scaffold, sometimes at least about 0.3 million cells / scaffold, sometimes at least about 0.5 million cells / scaffold, sometimes at least about 0.6 million cells / scaffold, sometimes at least about 0.7 million cells / scaffold, sometimes at least about 0.8 million cells / scaffold, sometimes at least about 0.9 million cells / scaffold, and sometimes at least about 1 million cells / scaffold.

[0402] In some examples, the cell density per scaffold ranges from about 0.1 million cells / scaffold to about 2 million cells / scaffold, sometimes from about 0.3 million cells / scaffold to about 1.5 million cells / scaffold, and sometimes from about 0.5 million cells / scaffold to about 1 million cells / scaffold.

[0403] The methods disclosed herein include inducing cell differentiation that grows on and / or within the pores of multiple scaffolds.

[0404] As used in this article, cell differentiation refers to the process by which cells undergo changes that allow them to specialize into various different cell types.

[0405] In the context of this disclosure, cells undergo differentiation on at least one surface of multiple scaffolds and / or within the pores of multiple scaffolds. The period during which cells are allowed to differentiate in a bioreactor is referred to herein as the differentiation phase. The differentiation phase follows the growth phase.

[0406] In some examples, the method includes conditions that support cell differentiation. In some examples, the method includes conditions that support the differentiation of at least a portion of the cells.

[0407] It is understandable that conditions during the differentiation phase are optimized to suit the cells undergoing differentiation in the bioreactor, and may vary depending on, for example, the cell type the cells will differentiate into.

[0408] In some examples, the conditions used during the growth phase are the same as those used during the differentiation phase. In some examples, one or more of the following—temperature, pH, dissolved oxygen (DO), agitation, osmotic pressure, and gas exchange—are the same during the growth phase as during the differentiation phase.

[0409] In some examples, the method involves perfusing the cavity with differentiation medium.

[0410] As described herein, cells grown on at least one surface of multiple scaffolds and / or within the pores of multiple scaffolds can differentiate into a large number of specialized differentiated cells. One of the conditions that influences the type of specialized differentiated cells is the differentiation culture medium.

[0411] Therefore, the choice of cell culture medium used during the differentiation phase may affect the type of specialized differentiated cells.

[0412] In some examples, the cell culture medium used during the differentiation phase is muscle differentiation medium.

[0413] As used in this article, muscle differentiation medium refers to a cell culture medium used to promote differentiation into muscle cells, particularly mature muscle cells or myotubes.

[0414] In some examples, the cell culture medium used during differentiation is adipocyte differentiation medium. As used herein, adipocyte differentiation medium refers to a cell culture medium used to promote differentiation into adipocytes. In some examples, the adipocyte differentiation medium contains one or more adipocyte differentiation inducers. In some examples, the adipocyte differentiation inducer includes one or more of insulin, dexamethasone, or 3-isobutyl-1-methylxanthine (IBMX). In some examples, the adipocyte differentiation medium contains both insulin and dexamethasone.

[0415] In some examples, the method includes inducing cells to undergo differentiation into one or more of adipocytes, endothelial cells, hematopoietic cells, myocytes, hepatocytes, chondrocytes, osteocytes, fibroblasts, or any combination thereof.

[0416] In some examples, the method involves inducing cells to undergo differentiation to form adipocytes.

[0417] In some examples, the method includes inducing cells to undergo differentiation to form one or more of adipose tissue, blood vessels, muscle tissue, liver tissue, cartilage tissue, bone tissue, connective tissue, or any combination thereof.

[0418] In some examples, the method involves inducing cells to undergo differentiation to form adipose tissue.

[0419] The method involves harvesting porous three-dimensional scaffold structures containing cells or tissues.

[0420] This method involves evaluating at least one parameter to determine the optimal time or conditions for cell harvesting. In some examples, the at least one parameter is at least one metabolite as described herein.

[0421] In some examples, the method includes harvesting during cell growth and before differentiation begins.

[0422] In some examples, the method involves harvesting undifferentiated cells.

[0423] In some examples, the method involves harvesting undifferentiated cells when the glucose consumption rate reaches a plateau.

[0424] In some examples, the method involves harvesting undifferentiated cells when the lactate formation rate reaches a stationary phase.

[0425] In some examples, the method involves harvesting undifferentiated cells when the NH3 formation rate reaches a stationary phase.

[0426] In some examples, the method involves harvesting undifferentiated cells when the LDH formation rate reaches a stationary phase.

[0427] In some examples, the method includes harvesting undifferentiated cells between 1 and 30 days post-inoculation, sometimes between 1 and 20 days, and sometimes between 1 and 10 days.

[0428] In some examples, the method includes harvesting undifferentiated cells 1 day after inoculation, sometimes 2 days after inoculation, sometimes 3 days after inoculation, sometimes 4 days after inoculation, sometimes 5 days after inoculation, sometimes 6 days after inoculation, sometimes 7 days after inoculation, sometimes 8 days after inoculation, sometimes 9 days after inoculation, sometimes 10 days after inoculation, sometimes 11 days after inoculation, sometimes 12 days after inoculation, sometimes 13 days after inoculation, sometimes 14 days after inoculation, sometimes 15 days after inoculation, sometimes 16 days after inoculation, sometimes 17 days after inoculation, sometimes 18 days after inoculation, sometimes 19 days after inoculation, sometimes 20 days after inoculation, sometimes 21 days after inoculation, sometimes 22 days after inoculation, sometimes 23 days after inoculation, sometimes 24 days after inoculation, sometimes 25 days after inoculation, sometimes 26 days after inoculation, sometimes 27 days after inoculation, sometimes 28 days after inoculation, sometimes 29 days after inoculation, and sometimes 30 days after inoculation.

[0429] In some examples, the method involves harvesting undifferentiated cells 7 days after inoculation.

[0430] In some examples, the method includes harvesting differentiated cells. In other examples, the method includes harvesting between 1 and 30 days after the start of perfusion with differentiation medium. In some examples, the method includes harvesting 1 day after the start of perfusion with differentiation medium, sometimes 2 days, sometimes 3 days, sometimes 4 days, sometimes 5 days, sometimes 6 days, sometimes 7 days, sometimes 8 days, sometimes 9 days, sometimes 10 days, sometimes 11 days, sometimes 12 days, sometimes 13 days, sometimes 14 days, sometimes 15 days, sometimes 16 days, sometimes 17 days, sometimes 18 days, sometimes 19 days, sometimes 20 days, sometimes 21 days, sometimes 22 days, sometimes 23 days, sometimes 24 days, sometimes 25 days, sometimes 26 days, sometimes 27 days, sometimes 28 days, sometimes 29 days, and sometimes 30 days after the start of perfusion with differentiation medium.

[0431] The method involves collecting harvested porous three-dimensional scaffold structures, on which at least growing cells are present.

[0432] In some examples, the method includes collecting a porous three-dimensional scaffold structure on which cellular tissue has grown. In some examples, at least a portion of the cells is embedded within the pores of the porous structure.

[0433] In some examples, the cells are undifferentiated cells. In some other examples, the cells contain different cell types. In some examples, the cells contain a single type of differentiated cell. In some examples, the cells contain more than one type of differentiated cell. In some examples, the cells contain adipocytes. In some examples, the cells contain muscle cells.

[0434] A porous three-dimensional scaffold structure on which cellular tissue has grown is collected by removing at least one end of the bioreactor.

[0435] This disclosure may cover a method in which cell seeding on multiple porous three-dimensional scaffold structures is performed outside a bioreactor, followed by the introduction of the scaffolds with attached cells into a packed bed bioreactor.

[0436] This disclosure also covers a method in which cells are seeded onto a scaffold outside a bioreactor (i.e., not inside the bioreactor). In other words, cells are brought into contact with the scaffold to allow cell seeding, and then the scaffold with the attached cells is packed into a packed bed bioreactor.

[0437] In some aspects, this disclosure provides a method comprising: (i) Introducing a cell culture medium containing cells attached to multiple porous three-dimensional scaffold structures into a packed bed bioreactor; the bioreactor includes a cavity 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 also including at least one fluid inlet at the first end and at least one fluid outlet. The internal space is configured to accommodate multiple porous three-dimensional support structures; Several of the porous three-dimensional scaffold structures contain edible materials; The three-dimensional scaffold structures are filled within the cavity, such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure; and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet. (ii) To provide conditions supporting cell growth and / or differentiation on a porous three-dimensional scaffold structure; and (iii) Harvest the porous three-dimensional scaffold structure with cells on it.

[0438] This disclosure also provides, according to some other aspects, an edible cultured cell cluster comprising a plurality of porous three-dimensional scaffold structures, each porous three-dimensional scaffold structure comprising edible material and at least cells carried by the edible material, the three-dimensional scaffold structures being distributed within the cell cluster such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure.

[0439] As described herein, porous three-dimensional scaffold structures are suitable for cell growth and / or cell differentiation. In some examples, multiple three-dimensional scaffold structures contain any one or a combination of undifferentiated cells, differentiated cells, extracellular matrix proteins, and cell-secreted metabolites adsorbed thereon.

[0440] In some examples, the edible cultured cell clusters are characterized by a protein content between about 0.5% and about 20% of the total wet weight of the cell clusters. In other examples, the edible cultured cell clusters are characterized by a protein content of about 0.5%, sometimes about 1%, sometimes about 2%, sometimes about 3%, sometimes about 4%, sometimes about 5%, sometimes about 6%, sometimes about 7%, sometimes about 8%, sometimes about 9%, sometimes about 10%, sometimes about 11%, sometimes about 12%, sometimes about 13%, sometimes about 14%, sometimes about 15%, sometimes about 16%, sometimes about 17%, sometimes about 18%, sometimes about 19%, and sometimes about 20% of the total wet weight of the cell clusters.

[0441] In some examples, the edible cultured cell clusters are characterized by a lipid content between about 0.1% and about 40% of the total wet weight of the cell clusters, sometimes between about 0.1% and about 30%, sometimes between about 0.3% and about 30%, sometimes between about 0.4% and about 25%, and sometimes between about 0.5% and about 20% of the total wet weight of the cell clusters.

[0442] In some examples, the edible cultured cell clusters are characterized by a lipid content of about 0.1%, sometimes about 0.3%, sometimes about 0.5%, sometimes about 0.7%, sometimes about 1%, sometimes about 3%, sometimes about 5%, sometimes about 7%, sometimes about 10%, sometimes about 13%, sometimes about 15%, sometimes about 17%, sometimes about 20%, sometimes about 23%, sometimes about 25%, sometimes about 27%, sometimes about 30%, sometimes about 33%, sometimes about 35%, sometimes about 37%, and sometimes about 40% of the total wet weight of the cell clusters.

[0443] In some examples, the edible cultured cell masses are characterized by a lipid-to-protein weight ratio between about 0.005 and about 80, sometimes between about 0.025 and about 40, and sometimes between about 0.1 and about 1. In some examples, the edible cultured cell masses are 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, or about 80.

[0444] In some examples, the edible cultured cell masses are characterized by a collagen to protein weight ratio between approximately 1% and approximately 50%. In other examples, the collagen to protein weight ratio is approximately 1%, sometimes approximately 2%, sometimes approximately 5%, sometimes approximately 7%, sometimes approximately 10%, sometimes approximately 15%, sometimes approximately 20%, sometimes approximately 25%, sometimes approximately 30%, sometimes approximately 35%, sometimes approximately 40%, sometimes approximately 45%, and sometimes approximately 50%.

[0445] like Figure 12C As shown, tissues grown in a bioreactor can be used to prepare food products.

[0446] Therefore, according to some other aspects, a food product is provided comprising an edible cultured cell cluster comprising a plurality of porous three-dimensional scaffold structures comprising edible material and cells carried at least on the edible material; the three-dimensional scaffold structures are distributed within the cell cluster such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure.

[0447] The food product is a cultured food product. In some examples, the food product is a cultured meat product.

[0448] This disclosure also provides a culture system, the culture system comprising: - A packed bed bioreactor, the packed bed bioreactor comprising a container 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 comprising at least one fluid inlet and at least one fluid outlet at the first end; The internal space is filled with multiple porous three-dimensional support structures; The multiple porous three-dimensional scaffold structures mentioned above contain edible materials. The three-dimensional scaffold structure is filled within the cavity such that each three-dimensional scaffold structure contacts at least one adjacent three-dimensional scaffold structure; and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet. - A cell culture medium reservoir in fluid communication with the internal space; - An oxygen source in fluid communication with the interior space; - A control module configured to control at least one parameter during operation of the bioreactor.

[0449] The cell culture medium reservoir is used to supply cell culture medium to at least one packed bed bioreactor.

[0450] In some examples, cell culture medium reservoirs contain cells.

[0451] In some examples, the cell culture medium reservoir contains growth medium.

[0452] In some examples, the cell culture medium reservoir contains differentiation medium.

[0453] In some examples, the cell culture medium reservoir contains oxygen dissolved in the growth medium and / or differentiation medium.

[0454] The cell culture medium reservoir is configured to supply cell culture medium to at least one packed bed bioreactor, the cell culture medium being configured to support the inoculation, growth and / or differentiation of cells and / or tissues as described herein, and is in fluid communication with the interior space of at least one bioreactor.

[0455] The term fluid connectivity refers to a device that allows fluid to flow between two components, such as pipes or conduits.

[0456] In some examples, the orientation of the cavity is positioned to allow fluid to flow upward from the first fluid inlet and through the packed bed.

[0457] In some examples, the cell culture medium is introduced via a first fluid inlet.

[0458] In some examples, the fluid outlet is located at the first end. In other examples, the fluid outlet is located at the second end.

[0459] The culture system may include additional components. In some examples, the culture system includes at least one port housing at least one sensor, as described herein. In some examples, the control module is configured to receive data from at least one sensor and operate the bioreactor based on the received data.

[0460] The culture system includes at least one pump.

[0461] In some examples, at least one pump is configured to control the flow rate of the culture medium flowing through the bioreactor.

[0462] In some examples, at least one pump is configured to direct fluid from a cell culture medium source and / or an oxygen source into the interior space.

[0463] As used herein, the term “about” means a value that may deviate from the mentioned value by a maximum of 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases by a maximum of 20%, including integer values ​​constituting a continuous range, and, where applicable, non-integer values. For example, the pH value described herein may be within ±0.5 or ±0.2 of the indicated value.

[0464] It should be noted that various embodiments of the present invention may be presented in a range format. A description of a range should be considered as having specifically disclosed all possible subranges, and the individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 or between 1 and 6 should be considered as having specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6.

[0465] It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims and their equivalents.

[0466] Throughout the specification and the subsequent claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" shall be understood to imply inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0467] It should be noted that, as used in this specification and the appended claims, the singular forms “an,” “a,” and “the” include plural references unless the context clearly specifies otherwise. For example, the term bioreactor may include one or more bioreactors having the described features. For example, the term scaffold may include one or more scaffolds, such as a plurality of similar scaffolds or a plurality of different scaffolds having the described features.

[0468] The following embodiments represent techniques employed by the inventors in carrying out various aspects of the invention. It should be understood that while these techniques are examples of preferred embodiments for practicing the invention, those skilled in the art will recognize, based on this disclosure, that many modifications can be made without departing from the spirit and scope of the invention.

[0469] It should be noted that the various embodiments and examples detailed herein in conjunction with various aspects of the invention are applicable to one or more aspects disclosed herein. It should be further noted that any embodiment described herein, such as those relating to food ingredient components, can be applied alone or in various combinations. The various embodiments and aspects of the invention described above and claimed in the following claims section are experimentally supported in the following examples.

[0470] The phrase "in another embodiment" as used herein, or any reference to an embodiment, does not necessarily refer to a different embodiment, although it may refer to different embodiments. Therefore, various embodiments of the invention may be combined (from the same or different aspects) without departing from the scope of the invention.

[0471] Non-limiting embodiments Example 1A: Preparation of different edible scaffolds The following describes the fabrication of different scaffolds.

[0472] Decellularization ( DC Edible support Edible scaffolds (carriers) are prepared by cutting different plants (including fruits, vegetables, roots, and stems) into scaffolds of different shapes (e.g., disc-shaped or hexagonal). Disc-shaped scaffolds are 6 mm in diameter and about 1 mm thick, while hexagonal scaffolds are 6 mm wide, 3.5 mm on each side, and 1 mm or 0.5 mm thick.

[0473] Template-based machine cutting of the support frame ensures uniformity and repeatability of the support frame geometry.

[0474] Next, the decellularization process was performed as follows: The scaffold was placed in SDS (sodium dodecyl sulfate, #75746, Sigma) solution and incubated with gentle shaking on an orbital shaker at room temperature for 48 hours. Subsequently, the scaffold was washed 3 to 5 times with deionized water (DW), then incubated in CaCl2 solution (#C1016, Sigma) on an orbital shaker at room temperature for 24 hours, followed by another DW wash. The cleaned scaffold was frozen at -20°C for 24 hours, then transferred to a deep freezer (-80°C) for at least overnight incubation. Finally, the scaffold was lyophilized for 48 hours and stored in a desiccator until use.

[0475] Tissue-processed plant protein ( TVP ) TVP, TSP, and TPP are sourced from Jinan DG, a Chinese company. TVP sheets (approximately 5mm thick) are precisely cut to the required size, for example, 1cm x 1cm. Before cell seeding, the scaffolds are sterilized using an autoclave and then thoroughly washed three times with PBS.

[0476] Gelatin-based scaffolds Various gelatin-based stents were purchased from Gelatex Technologies in Estonia, including #0601RN-SH and #compressed 0601RN-SH (used as...). Figure 2H and Figure 2I The scaffold is cut to the exact size required for the experiment (e.g., disc or hexagon). Before cell seeding, the scaffold is sterilized by autoclaving and then thoroughly washed three times with PBS.

[0477] Example 1B: Characterization of the water absorption capacity of the stent To characterize the water absorption capacity and porosity percentage of the stent, the dry and wet masses of the stent were weighed. The water retention capacity and stent porosity were then calculated.

[0478] result Examples of physical characteristics of hexagonal scaffolds in acellular plants: The resulting edible scaffold has a water retention capacity of approximately 97% and a porosity of 63%. This was determined using the following equation: water retention capacity of the support

[0479] Accordingly, the wet weight is approximately 20 mg / stent and the dry weight is approximately 0.6 mg / stent, providing approximately 97% (by weight) water retention capacity.

[0480] support porosity

[0481] The calculated total volume of the support is 0.306 cm³. 3 (Hexagonal, 6mm wide × 1mm thick), porosity 63%.

[0482] Example 2: Cell growth conditions in an edible packed bed (EPB) bioreactor Example 2A The effects of different scaffold materials on cell growth Materials and methods : Different types of edible scaffolds were prepared as described in Example 1 above. Three to five days before inoculation, the scaffolds were subjected to plasma treatment as described in Example 2C below. Next, as described in Example 2C below, the scaffolds were sterilized using a dry autoclave, followed by an coating step with an attachment solution.

[0483] avian fibroblasts ( Figures 2B to 2G ) or avian adult stem cells (pASC, isolation description see Example 4) Figure 2H and Figure 2ICells were seeded onto different scaffolds as follows: The cell pellet was suspended in growth medium (GM; - Growth medium: DMEM / F-12 (HIMEDIA, catalog number AT1184A), with 10% FBS (HyClone, catalog number SH30071.03), 1% GlutaMAX (Gibco, catalog number 35050-038), and 0.1% gentamicin (Gibco, catalog number 15750). The cell suspension was added to flasks containing a specific number of scaffolds (25 scaffolds correspond to 2.5 million cells = seeding density of 0.1M / scaffold) and gently shaken on a track shaker at 39°C for 3 to 4 hours. After 24 hours, the growth medium was changed, and the cells were allowed to grow on a track shaker at 38°C for 7 to 10 days, with the growth medium changed every 2 to 3 days.

[0484] To evaluate cell growth, an alpha blue assay was performed—samples from the cultured cell clusters were placed in growth medium containing 10% alpha blue solution (Biorad, catalog number BUF012B) and incubated at 39°C for 3 hours. The medium was then sampled, and signals were collected using a plate reader according to the company's protocol. Signal intensity was correlated with cell number. To visualize cell growth rate, signals at each time point were normalized to day 1.

[0485] Staining and Imaging – The cultured scaffolds were fixed in 4% paraformaldehyde (PFA) for 20–30 minutes and washed three times with PBS. Then, the scaffolds were placed in a PBS-prepared staining solution of 4',6-diamidinyl-2-phenylindole; Dapi (Sigma, catalog number D9542) (1:1000) and phalloidin (Sigma, catalog number P5282) (1:200) and stained on a rocker at room temperature for 1 hour. The scaffolds were then imaged using an LSM700 confocal microscope.

[0486] result : By culturing cells on different scaffolds, the ability of different scaffolds to support cell growth was tested, including a comparison of different plant-based scaffolds with animal-derived scaffolds (i.e., gelatin-based scaffolds).

[0487] like Figure 2A As shown, all plant-based scaffolds tested supported cell growth over time.

[0488] Figures 2B to 2I Confocal microscopy images of cells stained with Dapi and F-actin are shown, providing information about filamentous actin in the cell nucleus and cytoskeleton.

[0489] The results showed that all tested scaffolds, including those prepared from fungi, decellularized vegetable roots and stems, decellularized fruits, commercial TVP, commercial TSP, and gelatin-based scaffolds, supported cell growth and proliferation, demonstrating the versatility of the platform. These results indicate that different types of edible scaffolds, particularly plant-based scaffolds, are compatible with cell growth in the EPB bioreactor.

[0490] Example 2B The effects of different scaffold geometries and sizes on cell growth Edible scaffolds of different geometries were prepared as described in Example 1 above, and the bioreactor was filled with plasma-treated edible scaffolds.

[0491] The bioreactor was sterilized using a dry autoclave, while the pH and dissolved oxygen (DO) sensors were autoclaved separately and then aseptically placed into the bioreactor. The DO sensors were calibrated, and an attachment solution (-fibronectin attachment solution; Sartorius, catalog number 05-750-1H) was added to the bioreactor to allow cells to attach to an edible carrier. Primary avian adult stem cells (pASCs) suspended in cell growth medium (GM; as described in Example 2A) (isolation description in Example 4) were added to the bioreactor, and the cells attached to the scaffold and allowed to grow in the medium for 7 days while fresh medium was continuously supplied to the system via perfusion.

[0492] Sample the growth medium daily and check the glucose concentration. Calculate the glucose consumption rate (GCR) between the two samples using the following formula:

[0493] Q - Culture medium perfusion rate V - Bioreactor volume G in – Glucose concentration in fresh culture medium G0 – Glucose concentration in the first sample G f – Glucose concentration in the second sample Δt – Time difference between two samples BCA Total protein analysis (indicating protein content in culture products) was performed using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, catalog number 23225) according to the manufacturer's manual. In short, the sample was exposed to lysis buffer and homogenized by sonication. The sample was then centrifuged, the lysate was collected and mixed with the BSA working reagent, and then incubated for 30 minutes. The signal was then collected using a standard plate reader.

[0494] result : Cell growth was tested on different scaffolds with different geometries and sizes, including hexagonal and circular scaffolds (in...). Figures 3A to 3C The symbols are marked as "Hex" and "Disc".

[0495] Figure 3A The thickness of the support structure, measured from the top and bottom, is shown. It can be seen that two hexagonal supports with thicknesses of 1 mm and 0.5 mm, and a disc-shaped support with a thickness of 1.3 mm, were tested.

[0496] from Figure 3B It can be seen that the glucose consumption rate (GCR) was similar in the first few days of cell growth on different scaffolds. After 7 days of cell growth, a slight increase in GCR was observed in the hexagonal scaffold with a thickness of 0.5 mm.

[0497] exist Figure 3C The data shows the concentration of soluble proteins.

[0498] It should be noted that the amounts of GCR and soluble protein concentration measured on all scaffolds are comparable.

[0499] These results indicate that edible scaffolds of different geometries and sizes have been found to be compatible with cell growth in the EPB bioreactor.

[0500] Example 2C The effect of scaffold plasma treatment on cell growth method To evaluate the ability of plasma treatment to expose the cellulose structure in decellularized plant materials, an edible scaffold was prepared as described in Example 1 above.

[0501] Edible scaffolds were treated using a plasma apparatus – Vaculab, Tantec (2 mbar, 150 s). Both plasma-treated and untreated scaffolds were then immersed in 12 µM CBD-GFP solution provided by BioBetter. Samples were incubated at 4 °C for 3 h and then washed twice with PBS. GFP imaging of the samples was then performed using an LSM700 confocal microscope.

[0502] To evaluate the effects of plasma treatment on cell growth and protein production on decellularized plant scaffolds, plasma-treated and untreated scaffolds were incubated at room temperature in a fibronectin attachment solution (Sartorius, catalog number 05-750-1H, 1:50 dilution) for 1 hour.

[0503] pASC was then seeded onto the vector and incubated in cell growth medium at 37°C for 21 days, with the medium being changed every 2 to 3 days.

[0504] To evaluate cell growth, an alpha blue assay was performed (as described in Example 2A).

[0505] To evaluate the concentration of soluble protein, total BCA protein analysis was performed as described in Example 2B.

[0506] GFP imaging of the sample was performed using an LSM700 confocal microscope.

[0507] result The effects of plasma treatment on edible scaffolds Figure 4A and Figure 4B As shown in the figures, relatively low fluorescence intensity was observed in the untreated scaffold. Figure 4A ), while strong fluorescence intensity was observed in plasma-treated decellularized scaffolds ( Figure 4B ).

[0508] The results indicate that plasma treatment exposes the cellulose structure and may therefore facilitate cell attachment.

[0509] The effects of plasma treatment on cell growth Figure 4C As shown in the diagram, increased cell growth was observed in the plasma-treated scaffold. In addition to cell growth, other effects included... Figure 4D As shown, an increase in the amount of soluble proteins was observed in cells grown on plasma-treated scaffolds.

[0510] Example 2D The effect of the adhering solution on cell growth method To evaluate the effects of different attachment solutions on cell growth on decellularized plant scaffolds, scaffolds were prepared as described above and subjected to plasma treatment. The scaffolds were then incubated with one of the following solutions: 1. Adhesion solution - fibronectin adhesion solution (0.01 mg / ml) 2. Used culture medium - waste culture medium for culturing cells, composed of proteins and substances secreted by the cells.

[0511] 3. No adhesion – PBS only pASCs were then seeded onto the vector and incubated in cell growth medium at 37°C for 7 days. The number of viable cells on the scaffold was evaluated by alpha blue assay as described in Example 2A on days 1, 4, and 7.

[0512] As described in Example 2A, the sample was stained with Dapi and phalloidin.

[0513] result exist Figure 5A As can be seen, compared with cell growth without any solution, both tests showed increased cell growth with the attached solution.

[0514] Figures 5B to 5D Cell growth and proliferation are shown. These figures illustrate that treatment with solutions (attachment solution, used culture medium, and PBS) may be beneficial for cell growth and proliferation.

[0515] Example 3: Growth of different cells on edible scaffolds Materials and methods : Avian adult stem cells ( pASC Cells were isolated from 26-day-old Beijing duck embryos. The tissues were thoroughly washed with biological buffer, cut into small pieces, and enzymatically digested. The solution was then filtered through a 100µm filter, and the cells were centrifuged at 300g for 10 minutes. The cell pellet was resuspended in GM and then seeded into cell culture plates. Mycoplasma testing was performed throughout the isolation process.

[0516] -Bovine adult stem cells ( bASC Cells were isolated from adult bovine tissue. The tissue was thoroughly washed with water, cut open, and placed in a sterile bag containing an antimicrobial and antifungal agent. The isolation process was performed aseptically in a biosafety cabinet. The tissue was then minced, washed with biological buffer, and digested to release the cells. The isolated cells were seeded into tissue culture plates, and mycoplasma testing was performed at all stages of the isolation process. bASC growth medium: DMEM-high glucose (ATCC, Cat# 30-2002), supplemented with 10% FBS (Gibco, catalog number 19270-106) and 0.1% gentamicin (Gibco, catalog number 15750).

[0517] -Bovine dermal fibroblasts ( BDF Cells were purchased from the ScienCell research laboratory and isolated from fetal bovine skin. BDF growth medium: Fibroblast medium-2 (FM-2, catalog number 2331), supplemented with 5% FBS (catalog number 0025), 1% penicillin / streptomycin solution (P / S, catalog number 0503), and 1% fibroblast growth supplement-2 (FGS-2, catalog number 2382).

[0518] All cell types were cultured as previously described. After 7 days, the scaffolds were fixed with 4% paraformaldehyde (PFA) and F-actin and nuclei were stained with phalloidin and DAPI, respectively, as described in Example 2A. The scaffolds were then observed using a Zeiss LSM 700 confocal microscope.

[0519] result : from Figures 6A to 6H It can be seen that various cell types from different species have successfully grown on the edible scaffold. The edible scaffold also shows compatibility with avian and bovine cells, such as adult stem cells (ASCs) and fibroblasts.

[0520] Example 4: Characteristics of cell growth on edible scaffolds method To demonstrate cell growth within an edible packed bed, edible scaffolds of varying geometries were prepared as described in Example 1 above, and the bioreactor was filled with these plasma-treated edible scaffolds. The bioreactor was sterilized using a dry autoclave, while the pH and dissolved oxygen (DO) sensors were autoclaved separately and then aseptically placed into the bioreactor. The DO sensors were calibrated, and an attachment solution was filled into the bioreactor to allow cell attachment to the edible carriers.

[0521] Chicken embryonic fibroblasts (CEF; purchased from ATCC, catalog number CRL-3586) suspended in cell growth medium were added to the bioreactor. The cells attached to the scaffold and were allowed to grow in the medium for 10 days while fresh medium was continuously supplied to the system via perfusion.

[0522] After harvesting the biomass, the scaffold was separated, fixed in 4% PFA, and stained with Dapi and phalloidin as described in Example 2A. The scaffold was then imaged from both sides using an LSM700 confocal microscope to demonstrate cell cluster formation on both sides of the edible scaffold.

[0523] In addition, biomass was photographed to capture ECM formation between the scaffolds.

[0524] To demonstrate cell growth and tissue formation within the scaffold pores, duck embryonic fibroblasts ( DEF Cells (purchased from ATCC, catalog number CCL-141) were seeded onto decellularized plant scaffolds at a cell density of 20 k cells / scaffold. Cells were cultured for 14 days in DEF growth medium (EMEM (ATCC, catalog number 30-2003), supplemented with 10% FBS (Gibco, catalog number 19270-106) and 0.1% gentamicin (Gibco, catalog number 15750)). The resulting constructs were fixed in 4% PFA and then sectioned (5µm sections) and stained with Sirius red. The sections were then imaged using a Nikon inverted microscope Eclipse Ts2.

[0525] result The growth and distribution of CEF on the scaffold were tested. Figure 7A and Figure 7B These are confocal microscopy images of cells stained with Dapi and F-actin, taken from both sides (i.e., opposite sides) of the scaffold. As can be seen in these figures, a uniform distribution of cells is observed on all surfaces of the scaffold.

[0526] Figure 7C and Figure 7D The biomass obtained after CEF growth is shown, illustrating the growth of these cells within the scaffold.

[0527] Figures 7E to 7N Cross-sections of DEF throughout the test scaffold are shown, stained with Sirius red. These figures demonstrate that cell distribution within the scaffold was observed at all test depths.

[0528] The results showed that the scaffold supported the three-dimensional (3-D) growth of cells.

[0529] Example 5: Cell type growth in an edible packed bed bioreactor - For adipocyte differentiation, cells were induced with adipocyte differentiation medium: DMEM / F-12 (Sartorius, catalog number 01-170-1A), supplemented with 5% FBS (Sigma, catalog number F7524), 1% GlutaMAX (Gibco, catalog number 35050-038), 0.1% gentamicin (Gibco, catalog number 15750), insulin 20ug / ml (Merck, catalog number 16634), dexamethasone 1uM, oleic acid 40ug / ml (Sigma, catalog number O1383), linoleic acid 40ug / ml (Sigma, catalog number L1012), and D-glucose 4.5mg / ml (Sigma, catalog number G8769).

[0530] - Edible scaffolds are subjected to plasma treatment using a vacuum apparatus, and the bioreactor is filled with plasma-treated edible scaffolds (e.g., ...). Figure 5A (As shown). The bioreactor was sterilized using a dry autoclave, while the pH and dissolved oxygen (DO) sensors were autoclaved separately and then aseptically placed into the bioreactor. The DO sensors were calibrated, and an attachment solution was filled into the bioreactor to allow cells to attach to an edible carrier.

[0531] Cells suspended in cell growth medium were added to the bioreactor, attached to a scaffold, and allowed to grow in the medium for 7 days. On day 7, the medium was replaced with adipocyte differentiation medium to induce cell differentiation.

[0532] During the days of cell growth and differentiation in the packed bed bioreactor, the culture medium (growth or differentiation medium) was sampled daily, and the concentrations of glucose, lactate, ammonia, glutamine, and lactate dehydrogenase were quantified using a Cedex Bio analyzer. Metabolite consumption or formation rates were calculated and used to assess cell culture progress (for growth and / or differentiation).

[0533] The control experiments involved cell growth in Erlenmeyer flasks (“suspension”) and well plates (“direct plates”). Specifically, edible scaffolds were placed in Erlenmeyer flasks containing attachment medium, and then the attachment medium was replaced with cell-containing growth medium. Cells were allowed to grow for 7 days and then characterized, just as cells grown in a packed bed were. Additionally, edible scaffolds were placed in well plates, one scaffold per well, and attachment medium was added to each well. The attachment medium was then replaced with cell-containing growth medium, and cells were allowed to grow for 7 days and then characterized, just as cells grown in a packed bed were.

[0534] result Different parameters were monitored using enhanced GCR assays during cell growth and differentiation. Figures 8A to 8D Provides glucose consumption ( Figure 8A ), NH3 formation ( Figure 8B ), lactic acid formation rate ( Figure 8C ) and lactate dehydrogenase (LDH) form ( Figure 8D The chart shows the results. Data were collected almost daily during the 21-day period. The increase in all parameters during the cell growth phase (days 0 to 7) supports the conclusion that the edible scaffold supports cell growth in the EPB bioreactor. Tissue was harvested on day 21.

[0535] Example 6: Tissue Harvesting Materials and methods : - Almar blue assay (indicating cell density) – Samples from the cultured cell clusters were placed in growth medium containing 10% Almar Blue solution (Biorad, catalog number BUF012B) and incubated at 37°C for 3 hours. The medium was then sampled, and signals were collected using a plate reader according to the company's protocol.

[0536] - Collagen purple staining (indicator) ECM Formation and deposition ) - The edible carrier was fixed with 4% PFA and then embedded in paraffin. Lateral continuous paraffin sections of the scaffold (each 200 µm) were stained with Sirius red for collagen staining.

[0537] - LipidToxStaining (indicating triglyceride production in differentiated adipocytes) - Tissue was fixed with 4% PFA, washed three times with PBS, and then stained with HCS LipidTOX (ThermoFisher Scientific, catalog number H34475) according to the manufacturer's instructions.

[0538] - BCA Total protein analysis (indicating protein content in culture products) was performed using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, catalog number 23225) according to the manufacturer's manual. In short, the sample was exposed to lysis buffer and homogenized by sonication. The sample was then centrifuged, the lysate was collected and mixed with BSA working reagent, followed by incubation for 30 minutes. The signal was then collected using a standard plate reader.

[0539] - Triglyceride quantification (indicating triglycerides in culture products) – Performed using a triglyceride colorimetric assay kit (Cayman, catalog number 10010303) according to the manufacturer's manual. In short, the sample is exposed to lysis buffer and homogenized by sonication. The sample is then centrifuged, the lysate is collected and mixed with a triglyceride enzyme mixture. A colorimetric signal is then collected using a standard plate reader.

[0540] result Cultured tissues were characterized at the end of the growth phase (day 7) and the end of the differentiation phase (day 21). Specifically, collagen staining (performed to evaluate ECM deposition) and total protein analysis were performed at the end of the growth phase, while lipid staining and TG analysis were performed at the end of the differentiation phase.

[0541] To collect the edible scaffold with grown tissue, stop the control loop and disconnect the sensors, tubing, and rotator from the container. Place the container in a sterile environment and open it to access the filling bed. Remove the edible scaffold with the cell culture biomass (tissue) for subsequent analysis and integration into the final blend.

[0542] Characterization of tissues in harvested materials Figures 9A to 9H As shown in the image.

[0543] Figure 9A The images show cell nuclei and actin staining from tissues harvested after the growth period.

[0544] Figure 9B and Figure 9F The images show collagen staining (purple-red) and collagen analysis from tissues harvested after the growth period. Figure 9D and Figure 9EProtein analyses from this tissue are shown separately.

[0545] from Figure 9A It can be seen that during cell growth in the bioreactor, cells grow and proliferate, and as... Figure 9B As shown, collagen growth indicates the development of the extracellular matrix.

[0546] Protein content analysis (including collagen) Figures 9D to 9F The results showed that cells grown in a packed bed configuration had increased protein levels compared to cells grown in suspension in conical flasks or cell pellets (control).

[0547] Figure 9C The image shows LipidTOX staining (green) on tissues harvested 21 days after differentiation. Figure 9G and Figure 9H The triglyceride (TG) analysis from this tissue is shown separately.

[0548] from Figure 9C It can be seen that lipids are formed during differentiation, as shown by lipid droplet formation.

[0549] exist Figure 9G and Figure 9H As can be seen, the lipid content of tissues differentiating in the packed bed configuration was increased compared to cells undergoing suspension differentiation or cell sedimentation in the conical flask. The results indicate the presence of differentiation progression towards adipose tissue.

[0550] In addition, to evaluate the uniformity of tissue growth in the packed bed configuration, scaffold samples were taken from different regions (top and bottom) of the packed bed, and product uniformity analysis was performed as follows: The number of cells grown in EPB will be compared with the number of cells grown in well plates. Cell counts will be determined using an alpha blue assay. Figure 10A The results show that when using a packed bed configuration, significantly improved cell counts are achieved compared to "direct" (cells are injected directly onto the same scaffold in the well plate) or "suspension" (cells are suspended in the culture medium covering the scaffold and then attached to the same scaffold) seeding.

[0551] In addition, from Figure 10A It can be seen that cell growth is evenly distributed throughout the entire bed, as shown by the lack of significant differences between cell clusters sampled from the “top” and “bottom” regions of the bed.

[0552] like Figures 10B to 10D As shown, cell growth in all parts of the bioreactor is comparable.

[0553] Example 7: Myocyte Differentiation in an EPB Bioreactor method : To evaluate the compatibility of the EPB platform with myogenic differentiation, avian embryonic myoblasts (PEM) (provided by ProFuse, Israel) were seeded onto plasma-treated, edible, decellularized scaffolds (1 million cells / scaffold). Cells were cultured in proliferation medium at 37°C for 2 days, followed by either 2 days of myogenic differentiation medium or 2 days of proliferation medium (undifferentiated) (medium provided by ProFuse).

[0554] Staining and Imaging: Four days later, the scaffold was fixed with 4% paraformaldehyde (PFA) and stained with myosin heavy chain (MyHC) and DAPI. The scaffold was then imaged using a Zeiss LSM 700 confocal microscope.

[0555] The levels of MyHC protein separated by SDS-PAGE were detected using Western blotting. Protein extracts were electrophoresed on SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were then blocked with 5% skim milk and incubated with primary antibody at room temperature for 2–3 hours. After incubation with primary antibody, the membranes were washed three times for 5 minutes each. The membranes were then incubated with secondary antibody at room temperature for 1 hour, followed by three washes for 5 minutes each. Bands were detected using a gel scanner, and band intensity was quantified using densitometry. MyHC levels were normalized relative to GAPDH bands.

[0556] result : Figures 11A to 11B It is an image showing muscle formation after cell differentiation. Figure 11A As can be seen, the shape of slender, fibrous muscle cells (myotubes) is observed. Furthermore, Figure 11B The formation of multinucleated myotubes during differentiation is shown. These results indicate differentiation into myocytes.

[0557] Figure 11C The presence of MHC protein was observed only in differentiated cells, indicating differentiation into mature myofibrils. The quantification of data in... Figure 11D As shown in the image.

[0558] Example 8: Final Product Preparation result Figure 12A A representative packed-bed bioreactor is shown, comprising a cellulose-based disc-shaped scaffold made of decellularized scaffolds, cut into diameters of 6 mm and thicknesses of 1 mm, as described above, without any culture medium treatment or cell seeding. Figure 12B This is an image of the scaffold material harvested after the growth of adult stem cells, as described above. Cell growth was confirmed by Almarin assay.

[0559] Figure 12C Images of the cell attachment scaffold structure harvested after frying are provided. For sensory characteristics, the fried product was tested by a tasting panel, which confirmed that it possessed meat-like characteristics in appearance, texture, aroma, and taste.

[0560] from Figure 13 As can be seen, the exemplary culture pilot-scale system includes multiple packed-bed exemplary bioreactors (200), at least one fluid inlet (220) at the first end (240), and at least one fluid outlet (260).

[0561] Example 9: Cultivating the sensory effects of meat products To evaluate the sensory effects of cultured meat products in EPB, the cultured meat products were compared with cytoplasmic mixtures.

[0562] method : Samples with different weight percentages were prepared as follows: 1.96% growth medium and 4% scaffold (represented as 0% culture).

[0563] 2.89.2% growth medium, 4% scaffold, 6.8% cytoplasm (chicken embryonic fibroblasts). (This is expressed as 6.8% culture).

[0564] 3.46% growth medium, 4% scaffold, 50% cytoplasm (represented as 50% culture).

[0565] After mixing each sample until homogeneous, each sample was fried in a skillet with precise time and heat. All samples weighed the same (3 grams).

[0566] In addition, tissue samples grown in the EBP were fried in a pan under the same conditions. These samples were characterized by a cell content of 6.8% (grown on and within the scaffold).

[0567] Each fried sample was inserted into an opaque vial with a random number to make the test blind.

[0568] A group of 19 olfactory participants (each olfactory participant individually) were asked to rate the intensity of the chicken odor in each sample on the following scale: very low, low, medium, high, very high.

[0569] After each olfactory participant completes the sample scoring, all samples are reheated in vials for use by the next olfactory participant.

[0570] Each olfactory participant was asked to rate the sample relative to a fried chicken reference sample.

[0571] result : Figure 14 shows the average score for each sample, as well as the discreteness of each score. It can be seen that the cultured meat product prepared in EBP (containing 6.8% cells) is characterized by a higher sensory effect compared to the mixed product with the same percentage of cytoplasm.

[0572] Furthermore, the cultured meat product prepared in EBP (containing 6.8% cells) is characterized by having the same sensory effects as the mixed product consisting of 50% cytoplasm.

[0573] These results indicate that cell and tissue growth in EBP is advantageous because it provides desirable sensory properties at low cell density. This suggests that the increased cell production capacity and tissue yield in EBP are associated with the release of volatile compounds that mimic the sensory characteristics of conventional food products.

Claims

1. A packed bed bioreactor, the packed bed bioreactor comprising a cavity 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 comprising at least one fluid inlet at the first end and at least one fluid outlet; The internal space is filled with multiple porous three-dimensional support structures; The plurality of porous three-dimensional scaffold structures contain edible materials; The three-dimensional scaffold structure is filled in the cavity such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure. and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet.

2. The packed bed bioreactor according to claim 1, wherein the edible material comprises plant material.

3. The packed bed bioreactor according to claim 2, wherein the plant material is of fruit or vegetable origin.

4. The packed bed bioreactor according to any one of claims 1 to 3, wherein each porous three-dimensional scaffold structure in the porous three-dimensional scaffold structure is characterized by one or more of the following: (i) Includes at least one dimension with a size ranging from about 0.5 mm to about 500 mm. (ii) Contains pores with diameters between approximately 20 µm and approximately 800 µm. (iii) The porosity is in the range of approximately 50% to approximately 99%. (iv) Having a shape selected from the group consisting of: sheet-like, fibrous, bead-like, flaky, disc-shaped, spherical, cylindrical, ring-shaped, polygonal, and star-shaped.

5. The packed bed reactor according to any one of claims 1 to 4, wherein the packed bed reactor comprises at least one type of three-dimensional support structure.

6. The packed bed reactor according to any one of claims 1 to 4, wherein the packed bed reactor comprises at least two different types of three-dimensional support structures.

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

8. The packed bed reactor according to any one of claims 1 to 7, wherein the plurality of support structures are arranged in a uniformly mixed distribution.

9. The packed bed reactor according to any one of claims 1 to 7, wherein the plurality of support structures are arranged in a non-uniform, ordered distribution.

10. The packed bed reactor according to any one of claims 1 to 9, wherein the porous three-dimensional scaffold structure is pre-adjusted to promote cell adhesion to the scaffold structure.

11. The packed bed bioreactor of claim 10, wherein the pre-adjustment comprises any one of the following: plasma treatment or electrostatic charging of the surface of the porous three-dimensional scaffold structure; increasing the surface energy of the porous three-dimensional scaffold structure; removing lignin from the surface of the porous three-dimensional scaffold structure; or exposing cellulose residues on the surface of the porous three-dimensional scaffold structure.

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

13. The packed bed reactor of claim 12, wherein the 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, lactic acid, glutamine, glutamate, NH3 and lactate dehydrogenase (LDH), biomass weight, biomass level in the chamber, and foam.

14. The packed bed bioreactor according to any one of claims 1 to 13, wherein the orientation of the cavity is positioned to allow fluid to flow upward from the fluid inlet and through the packed bed.

15. The packed bed bioreactor according to any one of claims 1 to 14, wherein the packed bed bioreactor is used in conjunction with a culture medium source for culturing cells within the cavity.

16. A method, the method comprising: (i) Introducing a cell culture medium containing cells into a packed bed bioreactor, the packed bed bioreactor including a cavity 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. The internal space is filled with multiple porous three-dimensional support structures; The plurality of porous three-dimensional scaffold structures contain edible materials; The three-dimensional scaffold structure is filled in the cavity such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure. and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet. (ii) Providing conditions that support the growth of the cells on the porous three-dimensional scaffold structure; as well as (iii) Harvest the porous three-dimensional scaffold structure containing cells thereon.

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

18. The method of claim 16 or 17, the method comprising pre-adjusting the porous three-dimensional scaffold structure to promote cell adhesion to the scaffold structure.

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

20. The method according to any one of claims 16 to 19, the method comprising washing the porous three-dimensional scaffold structure with an adhering solution, wherein the washing is performed after the pre-adjustment if the pre-adjustment has been performed.

21. The method of claim 20, wherein the attachment solution is selected from the group consisting of: fresh culture medium, PBS, fibronectin attachment solution, collagen attachment solution, laminin attachment solution, vitrin attachment solution, elastin attachment solution, poly-L-lysine attachment solution, gelatin attachment solution, and used attachment solution.

22. The method according to any one of claims 16 to 21, wherein the cell culture medium comprises a single cell type or more than one cell type.

23. The method according to any one of claims 16 to 22, wherein the cell culture medium comprises at least stem cells.

24. The method according to any one of claims 16 to 23, wherein the cell culture medium 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 myoblasts, fibroblasts, hepatocytes, osteoblasts, chondrocytes, adipocytes, hepatocytes, osteoblasts, chondrocytes, and any combination thereof.

25. The method according to any one of claims 16 to 24, wherein the conditions supporting the growth of the cells on the porous three-dimensional scaffold structure include: (i) Temperatures between approximately 10°C and approximately 45°C (ii) pH in the range of approximately 6 to approximately 8, (iii) DO levels in the range of approximately 10% to approximately 100%, (iv) has a value between approximately 0.1 dyn / cm 2 With approximately 10 dyn / cm 2 Stirring of shear stress between them.

26. The method according to any one of claims 16 to 25, wherein the conditions supporting the growth of the cells on the porous three-dimensional scaffold structure include conditions supporting cell seeding and conditions supporting cell growth.

27. The method of claim 26, wherein the conditions include a flow rate between about 1 cm / s and 5 cm / s.

28. The method of claim 27, wherein the stirring during inoculation comprises stirring with alternating speeds, the alternating speeds comprising a first rotational speed in the range of 10 rpm to 50 rpm and a second rotational speed between 100 rpm and 200 rpm.

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

30. The method according to any one of claims 27 to 29, wherein the stirring during cell growth is performed at a rotational speed in the range of about 100 rpm to about 200 rpm.

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

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

33. The method according to any one of claims 16 to 32, wherein the cavity is perfused with growth medium and / or differentiation medium.

34. The method according to any one of claims 16 to 33, the method comprising providing the conditions supporting the growth of the cells.

35. The method according to any one of claims 16 to 34, the method comprising the conditions supporting the differentiation of at least a portion of the cells.

36. The method according to any one of claims 16 to 35, wherein the cells are undifferentiated cells and harvesting is carried out when the glucose consumption rate reaches a plateau.

37. The method of claim 36, wherein the cells comprise differentiated cells and are harvested between 1 and 30 days after the start of perfusion with differentiation medium.

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

39. The method according to any one of claims 16 to 38, the method comprising collecting harvested porous three-dimensional scaffold structures having growing cellular tissue thereon.

40. The method of claim 39, wherein at least a portion of the cell is embedded within the pores of the porous structure.

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

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

43. The edible cultured cell cluster according to claim 41 or 42, characterized in that... At least one of the following: - The protein content is between approximately 0.5% and 20% of the total wet weight of the cell cluster; - The lipid content is between approximately 0.1% and 40% of the total wet weight of the cell cluster; - The weight ratio of lipids to proteins is between approximately 0.1 and 1; - The weight ratio of collagen to protein is between approximately 1% and 50%.

44. A food product comprising an edible cultured cell cluster, the edible cultured cell cluster comprising a plurality of porous three-dimensional scaffold structures, the porous three-dimensional scaffold structures comprising edible material and cells carried on at least the edible material; the three-dimensional scaffold structures are distributed within the cell cluster such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure.

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

46. ​​A culture system, the culture system comprising: - A packed bed bioreactor, the packed bed bioreactor comprising a container 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 comprising at least one fluid inlet and at least one fluid outlet at the first end; The internal space is filled with multiple porous three-dimensional support structures; The multiple porous three-dimensional scaffold structures mentioned above contain edible materials. The three-dimensional scaffold structure is filled in the cavity such that each three-dimensional scaffold structure is in contact with at least one adjacent three-dimensional scaffold structure. and The porosity and distribution of the porous three-dimensional support structure within the internal space are selected to allow for substantially uniform fluid flow from the first fluid inlet to the fluid outlet. - A cell culture medium reservoir in fluid communication with the internal space; - An oxygen source in fluid communication with the interior space; - A control module configured to control at least one parameter during operation of the bioreactor.

47. The culture system of claim 46, wherein the orientation of the cavity is positioned to allow fluid to flow upward from the first fluid inlet and through the packed bed.

48. The culture system according to claim 46 or 47, wherein the fluid outlet is located at the second end.

49. The culture system according to any one of claims 46 to 48, the culture system comprising at least one port housing at least one sensor, and the control module being configured to receive data from the at least one sensor and operate the bioreactor based on the received data.

50. The culture system according to any one of claims 46 to 49, the culture system comprising at least one pump for directing fluid from the cell culture medium source and / or oxygen source into the internal space.

51. The culture system according to any one of claims 46 to 50, wherein the packed bed bioreactor is defined as any one of claims 1 to 15.

52. The culture system according to any one of claims 46 to 51, wherein the culture system is configured to perform the method according to any one of claims 36 to 40.

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