Edible meat artificially produced

专利摘要:

公开号:ES2685638T9
申请号:ES12817489T
申请日:2012-07-26
公开日:2019-02-04
发明作者:Gabor Forgacs；Francoise Marga；Karoly Robert Jakab
申请人:University of Missouri System；
IPC主号:

专利说明:

[0001] Edible meat artificially produced
[0002] Cross reference with related requests
[0003] This application claims the benefit of the U.S. Provisional Application. No. 61 / 511,948, filed on July 26, 2011.
[0004] BACKGROUND OF THE INVENTION
[0005] A protein is a nutrient that the human body needs for growth and maintenance. Apart from water, protein is the most abundant molecule in the body. According to the guidelines of the United States and Canada of reference food intake, women with an age of 19-70 years need to consume 46 grams of protein a day, while men with an age of 19-70 need to consume 56 grams of protein a day to avoid a lack. This recommendation, however, is for a sedentary person who has no disease. Lack of protein can lead to reduced intelligence or mental retardation, as well as contributing to the prevalence of diseases such as kwashiorkor. Protein deficiency is a serious problem in developing countries, especially in countries affected by war, hunger and overpopulation. Animal sources of protein, such as meat, are often a source of complete complement of all essential amino acids in adequate proportions.
[0006] The nutritional benefits of meat are mitigated by potentially associated environmental degradation. According to a 2006 report from the "Livestock, Environment and Development Initiative" initiative, entitled "Livestock's Long Shadow-Environmental Issues and Options," the livestock industry is one of the largest contributors to environmental degradation worldwide. , and the modern practices of raising animals for food, contributes greatly to air and water pollution, to soil degradation, climate change and the loss of biodiversity. The production and consumption of meat and other sources of animal protein are also associated with the cutting down of rainforests and the extinction of species. Accordingly, there is a need for an alternative solution to the demands of meat produced from live animals. In addition, Hopkins and Dacey, "Vegetarian Meat: Could Technology Save Animals and Satisfy Meat Eaters ", J Agric Environ Ethics (2008) 21: 579-596, propose that nascent tissue culture biotechnology, originally researched for medical applications, It is a promise for those who wish to eat meat, but not harm to animals because meat can be grown in vitro without killing animals.
[0007] Both Yang et al., "Cell sheet engineering: Recreating tissues without biodegradable scaffolds", Biomaterials (2005) 26: 6415-6422, and Matsuda et al., "Tissue Engineering Based on Cell Sheet Technology", Adv. Mater. (2007) 19: 3089-309999, describe the use of temperature-sensitive culture plates to achieve reversible adhesion of cells and detachment of plaque surfaces, allowing a non-invasive collection of cultured cells as a monolayer cell layer without the need for biodegradable structures or the use of proteolytic enzymes. By avoiding the use of any additional material, such as carrier or support substrates, the complications traditionally associated with artificial tissue production approaches, such as host inflammatory responses to implanted polymer materials, can be avoided. Therefore, the artificial production of cell layers allows a tissue regeneration through a direct transplantation of cell layers to host tissues or the creation of three-dimensional structures through the superposition of individual cell layers.
[0008] Sekine et al., "Myocardial tissue reconstruction: The cell sheet engineering approach", Inflammation and Regeneration (2007) 27 (3): 171-176, also propose an artificial production of tissues based on layers of cells, which implies stacking of cellular layers cultured in a confluent manner for the construction of dense 3-D cellular tissues. After superposition, the layers of individual cardiomyocytes are integrated to form a single tissue with continuous cell density, which resembles the natural heart muscle. When transplanted directly into host hearts, these artificially produced myocardial tissues are able to form morphological connections with the host, with the presence of functional slit-like junctions. Sekine et al. they have tried to favor neovascularization within artificially produced myocardial tissues to overcome the old limitations of artificially produced tissue thickness. As a possible advanced therapy, Sekine et al. they have tried to make functional myocardial tubes that may have the potential of circulatory support.
[0009] Aldhous, "Print me a heart and a set of arteries," New Scientist (15 April 2006) p.19, reported on bioprinting by using droplets of lumps of chicken heart cells that were a few hundred microns in size. diameter and they flowed together and were fused with alternating layers of support gel to form layers, rings or other forms of cells that appear to function normally.
[0010] In the U.S. patent application document 20050084958, Vein et al., Describe a meat product artificially produced with non-human tissue and a method for producing such a meat product. The meat product comprised muscle cells that were grown ex vivo and was used for the consumption of foods. The muscle cells could be cultured and fixed to a support structure and could be obtained from any non-human cell. The meat product could also comprise other cells such as fat cells or cartilage cells or both, which were cultured ex vivo together with the muscle cells.
[0011] In the U.S. patent application document 20100041134, Forgacs al., Describe structures and methods for the artificial production of tissues that include a multicellular body that includes a plurality of living cells. Forgacs et al. describe that a plurality of multicellular bodies can be arranged in a pattern and allowed to fuse to form an artificially produced tissue. The arrangement may include filler bodies that include a biocompatible material that resists migration and inward growth of the cells of the multicellular bodies and that resists adhesion of the cells thereto. Forgacs et al. disclose that three-dimensional artificial structures that can be assembled by printing or otherwise stacking the multicellular bodies and filler bodies, such that there is a direct contact between the contiguous multicellular bodies, suitably along a contact area that has a substantial length. The direct contact between the multicellular bodies favors an efficient and reliable fusion. The increase of the contact area between the adjacent multicellular bodies also favors an efficient and reliable fusion. Forgacs et al. they also describe production methods of multicellular bodies that have characteristics that facilitate the ability to assemble three-dimensional artificial structures.
[0012] Compendium of the invention
[0013] Artificial tissue production technology offers new opportunities to produce edible sources of animal protein that are not associated with the environmental degradation of livestock breeding. Artificial tissue production has been defined as an interdisciplinary field that applies the principles of artificial production and life sciences to the development of biological substitutes that restore, maintain or improve the function of a tissue or a complete organ. Langer R, Vacanti JP, Tissue Engineering, Science 260 (5110): 920-926 (May 1993). Despite the potential to apply the technology of artificial tissue production to meet the nutritional needs of living beings, scientifically and industrially viable procedures for producing edible meat have not been developed and artificially produced edible meat products are not available.
[0014] The invention provides a method for making artificially produced meat, as defined by the claims appended to this description.
[0015] A method for making artificially produced meat, wherein the method comprises:
[0016] preparing a plurality of multicellular bodies comprising a plurality of non-human myocytes coalesced with each other;
[0017] superimposing more than one multicellular body adjacently on a flat support substrate; fusing said multicellular bodies at least partially to each other to form a first layer; stack more than 50 additional layers on the first layer;
[0018] merge the stacked layers to form a volume of artificially produced meat; Y
[0019] cultivate the stacked layers to fuse the layers while the layers in an inner region of the volume die so that most of the cells in the volume are dead after the fusion between the layers is completed, at least partially, and
[0020] where artificially produced meat is edible.
[0021] As indicated above, the method of the invention optionally includes a step to prepare the plurality of multicellular bodies by culturing a plurality of non-human myocyte cells and non-human endothelial cells, at least until the cells have coalesced with each other . As mentioned above, any other appropriate non-human cell type can be included as part of some or all of the multicellular bodies that form the layers, including endothelial cells and / or fat cells and / or fibroblast cells.
[0022] During the formation of the artificially produced meat product, the layers can be stacked individually or collectively on top of another layer to create the volume of artificially produced meat. In some variations, each successive layer is oriented differently with respect to the adjacent layer (s). For example, as they are stacked, the new layers can be rotated relative to the other layers in the volume. In some variations, each layer is rotated approximately 90 ° relative to the other layers, when stacked.
[0023] In any artificially produced meat described herein, the layers can be exercised as they are formed. As described in more detail below, making the layers exercise can improve the formation of an extracellular matrix (ECM). This can also orient cells (eg, myocytes) within a layer as it is formed. Therefore, in some variations of the method to elaborate the meat produced artificially, it can be included a step of applying a mechanical, electrical or electromechanical force to exercise the myocytes in each layer.
[0024]
[0025] As mentioned, the step of stacking the layers includes stacking more than 50 layers, more than about 100 layers or the like.
[0026]
[0027] In some embodiments, the methods provided herein further comprise freezing said meat.
[0028]
[0029] In another aspect, methods for making artificially produced meat are disclosed herein, comprising: preparing a plurality of elongated multicellular bodies comprising a plurality of living non-human myocytes, wherein the cells adhere and / or cohere each; preparing a plurality of substantially spherical multicellular bodies comprising a plurality of living non-human myocytes wherein the cells adhere and / or are coherent with each other; overlaying more than one elongate multicellular body and more than one substantially spherical multicellular body adjacently on a support substrate; allowing said multicellular bodies to fuse to form a layer; superimpose (for example, stack) more than 50 layers on the first layer; allowing said layers to fuse to make artificially produced meat; and optionally, freezing said meat; provided that the artificially produced meat is edible and for ingestion. In some embodiments, methods for forming artificially produced meat are disclosed herein, comprising: preparing a plurality of elongated multicellular bodies comprising a plurality of non-human myocytes wherein the cells adhere and / or cohere each; preparing a plurality of substantially spherical multicellular bodies comprising a plurality of non-human myocytes wherein the cells adhere and / or are coherent with each other; overlaying more than one elongate multicellular body and more than one substantially spherical multicellular body adjacently on a support substrate; fusing said multicellular bodies to form a layer; superimpose more than 50 layers on the first layer; and fusing said layers to form a volume of artificially produced meat; provided that the artificially produced meat is edible and for ingestion. In some embodiments, the methods provided herein further comprise freezing said meat.
[0030]
[0031] The ratio of the elongated multicellular bodies to the substantially spherical multicellular bodies can be about 0: 100, 1: 100, 2: 100, 3: 100, 4: 100, 5: 100, 6: 100, 7: 100, 8 : 100, 9: 100, 1:10, 11: 100, 12: 100, 13: 100, 14: 100, 15: 100, 16: 100, 17: 100, 18: 100, 19: 100, 1: 5 , 21: 100, 22: 100, 23: 100, 24: 100, 25: 100, 26: 100, 27: 100, 28: 100, 29: 100, 3:10, 31: 100, 32: 100, 33 : 100, 34: 100, 35: 100, 36: 100, 37: 100, 38: 100, 39: 100, 2: 5, 41: 100, 42: 100, 43: 100, 44: 100, 45: 100 , 46: 100, 47: 100, 48: 100, 49: 100, 1: 2, 51: 100, 52: 100, 53: 100, 54: 100, 55: 100, 56: 100, 57: 100, 58 : 100, 59: 100, 3: 5, 61: 100, 62: 100, 63: 100, 64: 100, 65: 100, 66: 100, 67: 100, 68: 100, 69: 100, 7:10 , 71: 100, 72: 100, 73: 100, 74: 100, 75: 100, 76: 100, 77: 100, 78: 100, 79: 100, 4: 5, 81: 100, 82: 100, 83 : 100, 84: 100, 85: 100, 86: 100, 87: 100, 88: 100, 89: 100, 9:10, 91: 100, 92: 100, 93: 100, 94: 100, 95: 100 , 96: 100, 97: 100, 98: 100, 99: 100 or 1: 1. The ratio of the substantially spherical multicellular bodies to the elongated multicellular bodies can be about 0: 100, 1: 100, 2: 100, 3: 100, 4: 100, 5: 100, 6: 100, 7: 100, 8 : 100, 9: 100, 1:10, 11: 100, 12: 100, 13: 100, 14: 100, 15: 100, 16: 100, 17: 100, 18: 100, 19: 100, 1: 5 , 21: 100, 22: 100, 23: 100, 24: 100, 25: 100, 26: 100, 27: 100, 28: 100, 29: 100, 3:10, 31: 100, 32: 100, 33 : 100, 34: 100, 35: 100, 36: 100, 37: 100, 38: 100, 39: 100, 2: 5, 41: 100, 42: 100, 43: 100, 44: 100, 45: 100 , 46: 100, 47: 100, 48: 100, 49: 100, 1: 2, 51: 100, 52: 100, 53: 100, 54: 100, 55: 100, 56: 100, 57: 100, 58 : 100, 59: 100, 3: 5, 61: 100, 62: 100, 63: 100, 64: 100, 65: 100, 66: 100, 67: 100, 68: 100, 69: 100, 7:10 , 71: 100, 72: 100, 73: 100, 74: 100, 75: 100, 76: 100, 77: 100, 78: 100, 79: 100, 4: 5, 81: 100, 82: 100, 83 : 100, 84: 100, 85: 100, 86: 100, 87: 100, 88: 100, 89: 100, 9:10, 91: 100, 92: 100, 93: 100, 94: 100, 95: 100 , 96: 100, 97: 100, 98: 100, 99: 100 or 1: 1.
[0032]
[0033] In another aspect, methods for making artificially produced meat are disclosed herein, comprising: preparing a plurality of substantially spherical multicellular bodies comprising a plurality of living non-human myocytes, wherein the cells adhere and / or they cohere with each other; overlaying more than one substantially spherical multicellular body adjacently on a support substrate; allowing said substantially spherical multicellular bodies to fuse to form a layer; superimpose more than 50 layers on the first layer; allow the layers to fuse to form a volume of artificially produced meat; and optionally, freezing said meat; provided that the artificially produced meat is edible and for ingestion. In some embodiments, methods for making artificially produced meat are disclosed herein, comprising: preparing a plurality of substantially spherical multicellular bodies comprising a plurality of non-human myocytes wherein the cells adhere and / or cohere between yes; overlaying more than one substantially spherical multicellular body adjacently on a support substrate; fusing said substantially spherical multicellular bodies to form a layer; superimpose more than 50 layers on the first layer; and fusing said layers to form a volume of artificially produced meat; provided that the artificially produced meat is edible. In some embodiments, the methods provided herein further comprise freezing said meat.
[0034]
[0035] In some embodiments, the methods for making artificially produced meat described herein comprise the preparation of a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells adhere and / or are coherent with each other, wherein the multicellular bodies further comprise adipose cells and / or living non-human endothelial cells. In some embodiments, the multicellular bodies further comprise living, non-human fibroblast cells. In some embodiments, methods for making artificially produced meat, described herein, comprise overlaying more than one multicellular body adjacently on a support substrate, wherein the multicellular bodies are placed horizontally adjacently and / or vertically adjacently. . In some embodiments, the methods for making artificially produced meat, described herein, comprise layering more than one layer adjacently on a support substrate, wherein the layers are placed horizontally adjacently and / or vertically adjacently. In some embodiments, the support substrate is permeable to liquids and nutrients and allows the cell culture medium to come into contact with all surfaces of said multicellular bodies and / or layers. In some embodiments, the methods for making artificially produced meat, described herein, comprise allowing the multicellular bodies to fuse to form a layer, wherein the multicellular bodies fuse to form a layer in a cell culture environment. In some embodiments, the fusion of the multicellular bodies takes place from about 2 hours to about 36 hours. In some embodiments, the methods comprise allowing the layers to fuse to form artificially produced meat, wherein the layers are fused to form flesh artificially produced in a cell culture environment. In some embodiments, the melting of the layers is carried out for from about 2 hours to about 36 hours. In some embodiments, the elongated multicellular bodies of non-human myocytes and non-human endothelial cells have different lengths. In various embodiments, the elongated multicell bodies have a length of 1, 2, 3, 4, 5, 6, 7, 89 or 10 mm. In various embodiments, the elongated multicell bodies have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cm. In some embodiments, the elongated multicell bodies have a length ranging from 1 mm to 10 cm. In further embodiments, the elongated multicell bodies have a length ranging from about 1 cm to about 8 cm. In still other embodiments, the elongated multicell bodies have a length ranging from about 2 cm to about 6 cm. In some embodiments, methods for making artificially produced meat, described herein, comprise layering more than one layer adjacently on a support substrate and allowing the layers to fuse to make artificially produced meat. In various embodiments, the meat comprises 50, 60, 70, 80, 90 or 100 layers. In some embodiments, the methods for making artificially produced meat described herein comprise the preparation of a plurality of multicellular bodies comprising a plurality of living non-human myocytes, wherein the cells adhere and / or cohere with each other, wherein the multicellular bodies have a diameter adapted to allow diffusion to sufficiently sustain the maintenance and growth of non-human myocytes and non-human endothelial cells in culture. In various embodiments, the multicellular bodies have a diameter of 100, 200, 300, 400 or 500 μm. In some embodiments, the multicellular bodies have a diameter from 100 pma to 500 pM. In further embodiments, the multicellular bodies have a diameter from about 200 μm to about 400 μm. In some embodiments, the diameter is applied to the multicellular bodies substantially in the shape of a rod or sphere. In some embodiments, the methods for making artificially produced meat described herein comprise the preparation of a plurality of multicellular bodies comprising a plurality of living non-human myocytes wherein the cells adhere and / or are coherent with each other, wherein the multicellular bodies are bioprinted.
[0036] BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Fig. 1 represents a non-limiting example of a multicellular body; in this case, a multicellular body 1 with a width W1 that is approximately equal to the height H1 and a length L1 that is substantially greater than the width W1 or the height H1.
[0038] Fig. 2 represents a non-limiting example of a substantially spherical multicellular body; in this case, a substantially spherical multicellular body 2 with a width W1 that is approximately equal to the height H1.
[0039] Fig. 3 represents a non-limiting example of a multicellular body; in this case, a multicellular body 1 on a support substrate 3.
[0040] Fig. 4 represents a non-limiting example of a substantially spherical multicellular body; in this case, a substantially spherical multicellular body 2 on a support substrate 3.
[0041] Fig. 5 represents a non-limiting example of a method for preparing the multicellular bodies illustrated in Figs. 1-4; in this case, a method involving the transfer of a mixed cell pellet 4 to a capillary tube 5.
[0042] Fig. 6 represents a non-limiting example of a plurality of multicellular bodies; in this case, a plurality of multicellular bodies 1 is superimposed adjacently on a support substrate 3 in such a way that they are allowed to fuse.
[0043] Fig. 7 represents a non-limiting example of a plurality of substantially spherical multicellular bodies; in this case, a plurality of substantially spherical multicellular bodies 2 are superimposed adjacently on a support substrate 3 in such a way that they are allowed to fuse.
[0044] Fig. 8 represents a non-limiting example of a method for preparing a layer comprising a plurality of multicellular bodies; in this case, a method involving the extrusion of multicellular bodies 6 from a pressurized mechanical extruder comprising a capillary tube 5 on a support substrate 3.
[0045] Fig. 9 represents a non-limiting example of a method for preparing artificially produced meat; in this case, a method that involves superimposing more than one layer, comprising a plurality of multicellular bodies 7, 8, adjacently on a support substrate 3.
[0046] Fig. 10 represents a non-limiting example of a method for preparing artificially produced meat; in this case, a method that involves superimposing more than one layer, comprising a plurality of multicellular bodies 9 and a plurality of substantially spherical multicellular bodies 10, adjacently on a support substrate 3.
[0047] Fig. 11 represents a non-limiting example of a method for preparing artificially produced meat; in this case, a method that involves stacking more than one layer, where the layers after the first one are rotated 90 degrees with respect to the layer below.
[0048] Detailed description of the invention
[0049] Artificially produced tissue products prepared using traditional materials and methods are limited in size due to the short distances that gases and nutrients can diffuse to nourish the cells in the interior. In addition, existing techniques can not provide adequate speed and performance for the mass production of artificially produced products. As a result, existing methods of artificial tissue production, used to produce meat products, give rise to unattractive thin sheets and pastes, on a scale that is commercially unfeasible.
[0050] Therefore, an objective of the methods for the preparation of an edible meat product, described in this document, is to provide commercially viable and attractive meat products. Another objective is to provide high performance methods that are reliably, accurately and reproducibly improved to commercial levels. The advantages of the methods for preparing the edible meat products described in this document include, but are not limited to, the production of custom fabrics in a reproducible manner, with high performance and easily expandable, while maintaining precise control of the formation of a pattern, in particular in cases of multiple cell types, which can lead to artificially produced meat products with attractive taste, texture, thickness and appearance.
[0051] In this document, methods for making artificially produced meat are provided, in various embodiments, wherein the method comprises: a) preparing a plurality of multicellular bodies comprising a plurality of non-human myocytes that are coherent with each other; b) superimposing more than one multicellular body adjacently on a flat support substrate; c) fusing said multicellular bodies at least partially to each other to form a first layer; d) stacking more than 50 additional layers on the first layer; e) fusing the stacked layers to form a volume of artificially produced meat; and f) cultivate the stacked layers to fuse the layers, while the layers in the inner region of the volume die, so that most of the cells in the volume have died after the fusion between the layers is completed, at least partially , and where artificially produced meat is edible; and f) optionally freezing said meat; provided that the artificially produced meat is edible and for ingestion.
[0052] Also provided in this specification, in various embodiments, are methods of making artificially produced meat, comprising: a) preparing a plurality of elongated multicellular bodies and / or a plurality of substantially spherical multicellular bodies comprising a plurality of living non-human myocytes in where the cells adhere and / or cohere with each other; b) superimposing more than one elongated multicellular body and more than one substantially spherical multicellular body adjacently on a support substrate; c) allowing said multicellular bodies to fuse to form a layer; d) stacking more than 50 additional layers on the first layer; e) fusing the stacked layers to form a volume of artificially produced meat; and f) cultivate the stacked layers to fuse the layers while the layers in an inner region of the volume die, so that most of the cells in the volume are dead after the fusion between the layers is completed, at least partially, and where artificially produced meat is edible; and g) optionally freezing said meat; provided that the artificially produced meat is edible and for ingestion. Also described in this specification, in various embodiments, are methods for making artificially produced meat, comprising: a) preparing a plurality of elongated multicellular bodies and / or a plurality of substantially spherical multicellular bodies comprising a plurality of non-human myocytes wherein the cells adhere and / or cohere with each other; b) superimposing more than one elongated multicellular body and more than one substantially spherical multicellular body adjacently on a support substrate; c) fusing said multicellular bodies to form a layer; d) stacking more than one layer adjacent to each other on a support substrate; and e) fusing said layers to form a volume of artificially produced meat; provided that the artificially produced meat is edible. In some embodiments, the methods comprise overlaying more than one elongated multicellular body and more than one multicellular substantially spherical body in different relations of adjacent shape on a support substrate. In some embodiments, the methods further comprise freezing said meat.
[0053]
[0054] Also provided in this specification, in various embodiments, are methods of making artificially produced meat, comprising: a) preparing a plurality of substantially spherical multicellular bodies comprising a plurality of living non-human myocytes wherein the cells adhere and / or they cohere with each other; b) superimposing more than one substantially spherical multicellular body adjacently on a support substrate; c) allowing said substantially spherical multicellular bodies to fuse to form a layer; d) stacking more than about 50 additional layers on the first layer; e) fusing the stacked layers to form a volume of artificially produced meat; and f) cultivate the stacked layers to fuse the layers while the layers in an inner region of the volume die, so that most of the cells in the volume have died after the fusion between the layers is completed, at least partially, and where artificially produced meat is edible; and g) optionally freezing said meat; provided that the artificially produced meat is edible and for ingestion. Also described in this specification, in various embodiments, are methods for making artificially produced meat, comprising: a) preparing a plurality of substantially spherical multicellular bodies comprising a plurality of non-human myocytes, wherein the cells adhere and / or they cohere with each other; b) superimposing more than one substantially spherical multicellular body adjacently on a support substrate; c) fusing said substantially spherical multicellular bodies to form a layer; d) stacking more than about 50 additional layers on the first layer; e) fusing the stacked layers to form a volume of artificially produced meat; and f) cultivate the stacked layers to fuse the layers while the layers in an inner zone of the volume die, so that most of the cells in the volume are dead after the fusion between the layers is completed, at least partially, and where the artificially produced meat is edible. In some embodiments, the methods additionally comprise freezing said meat.
[0055]
[0056] A basic idea underlying classical artificial tissue production is to plant live cells in a biocompatible and, eventually, biodegradable structure, and then culture the system in a bioreactor so that the initial population of cells can expand into a tissue. The classical artificial production of fabrics has several shortcomings, especially when applied to the production of meat products. First, the method of seeding the cells generally involves contacting a solution of cells with a structure, such that the cells are trapped within the pores, fibers or other microstructure of the structure. This procedure is substantially randomized with respect to the placement of the cells within the structure and the placement of the cells relative to each other. Therefore, the sown structures are not immediately useful for the production of three-dimensional artificial structures that show patterns or a planned or predetermined placement of cells or aggregates of cells. Second, a selection of the biomaterial structure that is ideal for a given cell type is problematic and is often done by trial and error. Even if the right biomaterial is available, a structure can interfere with the achievement of high cell density. On the other hand, artificial fabric production based on structures does not increase easily or reliably up to industrial levels.
[0057]
[0058] In some embodiments, artificially produced meat products, layers and multicellular bodies are prepared with a method that utilizes a rapid prototyping technology based on a three-dimensional, automated, computer-assisted deposit of the multicellular bodies (eg. example, cylinders and spheroids) and a biocompatible support structure (eg, composed of agarose base) by a three-dimensional delivery device (eg, a bioprinter). The term "artificial production" means normally produced by man or arranged when used to refer to the layers and meat products described herein. An example of an artificially produced meat may include the arrangement or placement of multicellular bodies and / or layers to make the meat products artificially produced by a computer-aided device (eg, a bioprinter) in accordance with a computer command sequence. In other embodiments, the computer script is, for example, one or more computer programs, computer applications or computer modules. In still other embodiments, three-dimensional tissue structures are made through a post-impression fusion of multicellular bodies in a manner similar to self-assembly phenomena in early morphogenesis.
[0059] Unlike other artificially produced tissues, the artificially produced meat described herein is formed by a stack of layers of two-dimensional flat sheets of multicellular bodies at least partially fused. Therefore, the methods for making even large volumes of artificially produced meat, described in this document, may not have to simultaneously require the formation of three dimensional patterns, but may be done by a (parallel) cultivation of multiple two-dimensional layers that can be then assemble into a three-dimensional set or subsets that can then be stacked together. This advantageous method for making the artificially produced meats described herein, may allow the volume of artificially produced meat to be formed without having the need for three-dimensional support frames or structures, such as filling bodies. In addition, the two-dimensional layers can be formed in parallel with a relatively thin thickness that allows a diffusion of the nutrients from a culture medium in the flat layer during cultivation (for example, during the melting of the multicellular bodies of the components inside). of the layer). Only after stacking the layers of components to form the volume, then the diffusion of the nutrients can be limiting, giving rise to the cell death
[0060] Therefore, although a number of methods are available for arranging the multicellular bodies on a support substrate to produce a three-dimensional structure including manual placement, including positioning by an automated, computer-assisted machine, such as a bioprinter, such Methods may be useful, but they are not necessary. The advantages of a delivery of the multicellular bodies with bioprinting technology include a rapid, accurate and reproducible placement of the multicellular bodies to produce artificial structures that show planned or predetermined orientations or patterns of multicellular bodies and / or layers with various compositions. The advantages also include ensuring high cell density, while minimizing cellular damage frequently associated with other deposit methods based on solid free-form manufacturing, focused on cell printing in combination with hydrogels.
[0061] Methods for preparing or processing artificially produced meats, and commercial methods are also described herein. In some embodiments, the speed and ability to extend the techniques and methods described herein are used to design, construct and operate industrial and / or commercial facilities for the production of artificially produced edible meat products. In additional embodiments, artificially produced meat products are produced, packaged, frozen, stored, distributed, marketed, promoted and sold as, for example, food products for humans, components or ingredients of food products for humans, food products for animals. non-human or components or ingredients of food products for non-human animals.
[0062] Cells
[0063] Many types of self-adhering cells can be used to make the multicellular bodies, layers and artificially produced meat products described herein. In some embodiments, the artificially produced meat products are designed to resemble traditional meat products and the cell types are chosen to approximate those found in traditional meat products. In additional embodiments, artificially produced meat products, layers and multicellular bodies include non-human myocytes. In still other embodiments, artificially produced meat products, layers and multicellular bodies include non-human myocytes, and / or endothelial cells, and / or fat cells, and / or fibroblasts.
[0064] In general, the artificially produced meats described herein may differ from natural meats and other meats artificially produced by lack of blood vessels, and also lack nerve innervation. Even in variations in which endothelial cells are included as a component of one or more multicellular bodies, artificially produced meat will not include blood vessels competent to transmit blood. Therefore, even the large volumes of artificially produced meat made by the methods described herein, may not have any blood vessels. In addition, the artificially produced meats described herein may lack any nervous component (e.g., axons, dendrites, nerve cell bodies), since they may be grown without such components.
[0065] Humans traditionally eat various types of animal muscle tissue. Therefore, in some embodiments, the myocytes are skeletal myocytes. In some embodiments, the myocytes are cardiac myocytes. In some embodiments, the myocytes are smooth myocytes. In some embodiments, the endothelial cells are microvascular endothelial cells.
[0066] In other embodiments, artificially produced meat products include neural cells, connective tissue (including bone, cartilage, cells that differentiate into bone-forming cells and chondrocytes, and lymphatic tissues), epithelial cells (including endothelial cells that form the coatings in cavities and vessels or channels, exocrine secretory epithelial cells, absorbent epithelial cells, keratinizing epithelial cells and extracellular matrix secreting cells), and undifferentiated cells (such as embryonic cells, stem cells and other precursor cells), among others.
[0067] In some embodiments, the cells used to make a multicellular body are obtained from a living animal and cultured as a primary cell line. For example, in additional embodiments, the cells are obtained by biopsy and are cultured ex vivo. In other embodiments, the cells are obtained from commercial sources.
[0068] The artificially produced meat products and the layers comprising a plurality of multicellular bodies for use in the production of said meat described herein, are edible and are intended for consumption by humans, non-human animals or both. In some embodiments, the artificially produced meat products are human food products. In other embodiments, the artificially produced meat products are an animal feed, such as a feed for livestock, feed for aquaculture or pet food. Therefore, in view of the description provided herein, those skilled in the art will recognize that non-human cells from a variety of sources are suitable for use in the production of such products and with the methods described herein. document. In various embodiments, the multicellular bodies, the layers comprising the bodies multicellular and artificially produced meat products comprise non-human cells obtained from, by way of non-limiting examples, mammals, birds, reptiles, fish, crustaceans, molluscs, cephalopods, insects, non-arthropod invertebrates and combinations thereof.
[0069]
[0070] In some embodiments, suitable cells are obtained from mammals such as antelope, bear, beaver, bison, boar, camel, caribou, cow, deer, elephant, elk, fox, giraffe, goat, hare, horse, mountain goat, kangaroo, lion, llama, american moose, saino, pig, rabbit, seal, sheep, squirrel, tiger, whale, yak and zebra or combinations thereof. In some embodiments, suitable cells are obtained from birds such as chicken, duck, emu, goose, capercaillie, ostrich, pheasant, pigeon, quail and turkey or combinations thereof. In some embodiments, suitable cells are obtained from reptiles, such as turtle, snake, crocodile and alligator or combinations thereof. In some embodiments, suitable cells are obtained from fish such as anchovy, sea bass, catfish, cod, eel, plaice, fugu, grouper, haddock, halibut, herring, mackerel, lampugo, marlin, emperor, perch, pike. , haddock, salmon, sardine, shark, snapper, sole, swordfish, tilapia, trout, tuna and walleye or combinations thereof. In some embodiments, suitable cells are obtained from crustaceans such as crab, crayfish, lobster, shrimp and shrimp or combinations thereof. In some embodiments, suitable cells are obtained from molluscs such as abalone, clam, shell, mussel, oyster, scallop and snail or combinations thereof. In some embodiments, suitable cells are obtained from cephalopods such as cuttlefish, octopus and squid or combinations thereof. In some embodiments, suitable cells are obtained from insects such as ant, bee, beetle, butterfly, cockroach, cricket, damselfly, dragonfly, earwig, flea, fly, grasshopper, mantis, ephemeral, moth, silverfish, termite. , wasp or combinations thereof. In some embodiments, suitable cells are obtained from non-arthropod invertebrates (e.g., worms) such as flatworms, tapeworms, trematodes, trichinae, roundworms, hookworms, segmented worms (e.g., worms, polychaetes, etc.) or combinations of them.
[0071]
[0072] Multicellular organisms
[0073]
[0074] In this document, multicellular bodies are described that include a plurality of non-human living cells in which the cells adhere and / or cohere with each other. Also described herein are methods comprising: preparing a plurality of multicellular bodies comprising a plurality of living non-human myocytes, wherein the cells adhere and / or are coherent with each other; superimposing more than one multicellular body adjacently on a support substrate; and allowing the multicellular bodies to fuse to form a substantially flat layer for use in making artificially produced meat. In some embodiments, a multicellular body comprises a plurality of cells adhered and / or bonded together in a desired three-dimensional shape with viscoelastic consistency and sufficient integrity to facilitate handling and handling during an artificial bioproduction process, such as an artificial production of tissues. In some embodiments, sufficient integrity means that the multicellular body, during subsequent handling, is able to retain its physical form, which is not rigid, but which has a viscoelastic consistency, and to maintain the vitality of the cells.
[0075]
[0076] In some embodiments, a multicellular body is homocellular. In other embodiments, a multicellular body is heterocellular. In homocellular multicellular bodies, the plurality of living cells includes a plurality of living cells of a single cell type. Substantially, all living cells in a homocellular multicellular body are substantially cells of a single cell type. In contrast, a heterocellular multicellular body includes a significant number of cells of more than one cell type. Living cells in a heterocellular body can remain unclassified or can be "sorted" (eg, self-assembled) during the fusion process to form a particular internal structure for the artificially produced tissue. The ordering of the cells is consistent with the predictions of the differential adhesion hypothesis (DAH, from the English "Differential Adhesion Hypothesis"). The DAH explains the liquid-like behavior of cell populations in terms of tissue and interfacial surface tensions, generated by adhesive and cohesive interactions between cellular components. In general, the cells can be ordered based on differences in the adhesive forces of the cells. For example, the types of cells that are arrayed into a heterocellular multicellular body generally have a stronger adhesion resistance (and therefore a higher surface tension) than cells that are arranged on the outside of the body multicellular.
[0077]
[0078] In some embodiments, the multicellular bodies described herein also include one or more components of an extracellular matrix (ECM) or one or more derivatives of one or more components of the ECM, in addition to the plurality of cells. For example, multicellular bodies may contain various ECM proteins including, by way of non-limiting examples, gelatin, fibrinogen, fibrin, collagen, fibronectin, laminin, elastin and proteoglycans. The components of the ECM or the derivatives of the ECM components can be added to a cell paste used to create a multicellular body. The components of the ECM or derivatives of the ECM components added to a cell paste can be purified from an animal source or be produced by recombinant methods known in the art. Alternatively, the components of the ECM or the derivatives of the ECM components can be secreted naturally by the cells in the multicellular body.
[0079]
[0080] In some embodiments, a multicellular body includes a tissue culture medium. In additional embodiments, the Tissue culture medium can be any physiologically compatible medium and is usually selected according to the type or types of cells involved, as is known in the art. In some cases, the appropriate tissue culture medium comprises, for example, basic nutrients, such as sugars and amino acids, growth factors, antibiotics (to minimize contamination), etc.
[0081] Adhesion and / or cohesion of the cells in a multicellular body is suitably strong enough to allow the multicellular body to retain a three-dimensional shape while supporting itself on a flat surface. For example, in some cases, a multicellular body that attaches itself to a flat substrate may exhibit some minor deformation (eg, where the multicellular body comes into contact with the surface), however, the body multicellular is sufficiently cohesive to retain a height that is at least half its width, and in some cases, approximately equal to the width. In some embodiments, two or more multicellular bodies placed in an adjoining relationship side by side, facing each other on a flat substrate, form an empty space below their sides and above the work surface. See, for example, Figs. 3 and 4. In further embodiments, the cohesion of the cells in a multicellular body is strong enough to allow the multicellular body to support the weight of at least one multicellular body of similar size and shape, when the multicellular body is assembled to form an artificial structure in which the multicellular bodies are stacked one on top of the other. See, for example, Figs. 9 and 10. In still other embodiments, the adhesion and / or cohesion of the cells in a multicellular body is also suitably strong enough to allow the multicellular body to be picked up with an instrument (e.g., a micropipette). capillary).
[0082] In view of the description provided herein, those skilled in the art will recognize that multicell bodies having different sizes and shapes are within the scope of the embodiments provided herein. In some embodiments, a multicellular body is substantially cylindrical and has a substantially circular cross section. For example, a multicellular body, in various embodiments, has an elongated shape (e.g., a cylindrical shape) with a square, rectangular, triangular, or any other non-circular shape. Similarly, in various embodiments, a multicellular body has a generally spherical shape, a non-elongated cylindrical shape or a cuboidal shape. In various embodiments, the diameter of a multicellular body is approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 , 1000 | jm or quantifiable increases in them. In some embodiments, a multicellular body is configured to limit cellular necrosis caused by the inability of oxygen and / or nutrients to diffuse to central portions of the multicellular body. For example, a multicellular body is conveniently configured such that none of the living cells therein is more than about 250 jm from an outer surface of the multicellular body, and more suitably so that none of the living cells therein it is more than about 200 jm from the outside of the multicellular body.
[0083] In some embodiments, the multicellular bodies have different lengths. In other embodiments, the multicellular bodies have substantially similar lengths. In various embodiments, the length of a multicellular body is 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5, 5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 mm or quantifiable increases therein. In other various embodiments, the length of a multicellular body is 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5 , 5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 cm or quantifiable increments therein. In some embodiments, the length of the multicellular bodies is chosen so as to result in a shape and / or size of artificially produced meat product that approximates that of a traditional meat product, eg, a bacon strip, a hamburger , a fish fillet, a chicken breast or a steak.
[0084] Referring to FIG. 1, in some embodiments, a multicellular body 1 is substantially cylindrical with a width W1 approximately equal to a height H1 and has a substantially circular cross-section. In further embodiments, a multicellular body 1 is elongated with a length L1. In still other embodiments, W1 and H1 are suitably from about 300 to about 600 jm and L1 is suitably from about 2 cm to about 6 cm.
[0085] Referring to FIG. 2, in some embodiments, a multicellular body 2 is substantially spherical with a width W1 approximately equal to a height H1. In further embodiments, W1 and H1 are suitably from about 300 to about 600 jm.
[0086] Layers
[0087] The artificially produced meat obtained by the method according to the invention includes more than 50 layers on the first layer, wherein each layer includes a plurality of multicellular bodies comprising a plurality of non-human living cells, wherein the cells adhere and / or they are cohesion with each other. Also described herein are methods comprising the steps for superposing multicellular bodies adjacently on a support substrate and allowing the multicellular bodies to fuse to form a substantially planar layer for use in the formation of artificially produced edible meat products. In some embodiments, each layer is bioprinted, using the techniques described herein.
[0088] In some embodiments, a layer includes homocellular multicellular bodies. In other embodiments, a layer includes heterocellular multicellular bodies. In still other embodiments, a layer includes both multicellular and homocellular as well as heterocellular bodies. In additional embodiments, one layer includes non-human myocytes. In yet other embodiments, a layer includes non-human myocytes, non-human endothelial cells and adipose cells and / or fibroblast cells. In yet other embodiments, a layer includes non-human myocytes, non-human endothelial cells, and other cell types described herein.
[0089] In embodiments that include both non-human myocytes and non-human endothelial cells, a layer can include non-human myocytes and non-human endothelial cells in a ratio of about 30: 1, 29: 1, 28: 1, 27: 1, 26: 1 25: 1, 24: 1, 23: 1, 22: 1, 21: 1, 20: 1, 19: 1, 18: 1, 17: 1, 16: 1, 15: 1, 14: 1, 13 : 1, 12: 1, 11: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1 and 1: 1 or increases in them. In some embodiments, a layer contains non-human myocytes and non-human endothelial cells in a ratio of from about 19: 1 to about 3: 1. In various embodiments, one layer includes non-human endothelial cells comprising about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% and 25% or increases thereof, of the total cellular population. In some embodiments, a layer includes non-human endothelial cells comprising from about 5% to about 15% of the total cell population. In additional embodiments, the presence of endothelial cells contributes to endothelialization, described further herein.
[0090] In various embodiments, the thickness of each layer is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000 or 5000 | jm or quantifiable increases in them. In some embodiments, the thickness of each layer is chosen to allow diffusion to sufficiently support the maintenance and growth of substantially all cells in the culture layer.
[0091] In various embodiments, the plurality of layers includes approximately 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or 500 layers or increases in them. In some embodiments, the number of layers is chosen to result in an artificially produced meat product having a thickness approaching that of a traditional meat product, eg, a strip of bacon, a hamburger, a fish fillet, a breast of chicken or a steak.
[0092] In some embodiments, the artificially produced layers are designed to resemble traditional meat products and the design parameters (e.g., cell types, additives, size, shape, etc.) are chosen to approximate those found in traditional meat products. In additional embodiments, a layer is characterized by a nutritional composition that is substantially similar to traditional meat products. In yet other embodiments, a layer is characterized by a nutritional composition that is substantially in a 60-80 percent aqueous fluid, 14-35 percent protein, 1-25 percent fat, 1-5 percent carbohydrate and 1-5 percent other substances. In some embodiments, the myocytes of the artificially produced layers or the endothelized meat are aligned. In some embodiments, the myocytes are aligned by the application of an electric field as is known in the art. In some embodiments, the myocytes are aligned by the application of a mechanical stimulus, such as a cyclic stretch and relaxation of the substrate, as is known in the art. In further embodiments, aligned myocytes (eg, electro-oriented and mechano-oriented) have substantially the same orientation in relation to one another as that found in many animal muscle tissues. In some embodiments, the layers of the multicellular bodies provided herein are exposed to electrical and / or mechanical stimulation to facilitate the formation of a physiological disposition of the muscle cells.
[0093] Additives
[0094] In some embodiments, artificially produced meat products, artificially produced layers and / or multicellular bodies include one or more nutritional supplements. In additional embodiments, one or more nutritional supplements are selected from: vitamins, minerals, fiber, fatty acids and amino acids. In some embodiments, artificially produced meat products, layers and / or multicellular bodies include one or more additives to enhance commercial appeal (e.g., appearance, taste, color, odor, etc.). In further embodiments, the products of artificially produced meat, the layers and / or the multicellular bodies include one or more flavoring agents, one or more colorants and / or one or more odoriferous agents.
[0095] In some embodiments, the products of artificially produced meat, the artificially produced layers and / or the multicellular bodies include one or more from: matrix proteins, proteoglycans, antioxidants, perfluorocarbons and growth factors. The term "growth factor", as used herein, refers to a protein, polypeptide or complex of polypeptides, including cytokines, which are produced by a cell and which may affect itself and / or to a variety of other neighboring or distant cells. Typically, growth factors affect the growth and / or differentiation of specific cell types, either in development or in response to a multitude of physiological or environmental stimuli. Some, but not all growth factors are hormones. Exemplary growth factors are insulin, insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factors (FGFs), including basic FGF (bFGF), platelet-derived growth factors (PDGFs), including PDGF-AA and PDGF-AB, growth factor of hepatocytes (HGF), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-p), including TGFpl and TGFp3, epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor ( GM-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 6 (IL-6), IL-8 and the like.
[0096] In some embodiments, artificially produced meat products, artificially produced layers and / or multicellular bodies include one or more food preservatives known in the art. In some embodiments, the preservatives are antimicrobial preservatives which include, by way of non-limiting examples, calcium propionate, sodium nitrate, sodium nitrite, sulfites (eg, sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc.). .) and disodium ethylenediaminetetraacetic acid (EDTA). In some embodiments, the preservatives are antioxidant preservatives including, by way of non-limiting examples, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).
[0097] Substrate support
[0098] In the present document, a plurality of multicellular bodies disposed adjacently on a support substrate to form a substantially planar layer for use in the manufacture of artificially produced edible meat is described in some embodiments. Also described herein, in some embodiments, are methods comprising arranging the multicellular bodies adjacently on a support substrate to form substantially flat layers, superimposing more than one layer adjacently on an individual support substrate and allowing the layers are fused to make artificially produced meat. For example, a plurality of layers can be formed as described above, at the same time on different substrates, then removed from their substrate when the multicellular bodies have been fused sufficiently so that they can be removed and stacked on top of each other , or on top of a single substrate. In general, each layer includes non-human myocytes. Cells in the central portions of such artificial structures are normally supplied with oxygen and nutrients by diffusion; however, gases and nutrients normally diffuse approximately 200-300 pm into three-dimensional artificial cellular structures.
[0099] In some embodiments, the multicellular bodies described herein have a diameter adapted to allow diffusion to sufficiently promote maintenance and growth of said non-human myocytes in culture. As a result, in additional embodiments, the layers described herein have a thickness adapted to allow diffusion to sufficiently promote the maintenance and growth of said non-human myocytes in culture.
[0100] To facilitate and improve diffusion, in some embodiments, a support substrate is permeable to fluids, gases and nutrients, and allows the cell culture medium to contact all the surfaces of the multicellular bodies and / or the layers. during, for example, growth, maturation and fusion. In various embodiments, a support substrate is prepared based on natural biomaterials, synthetic biomaterials and combinations thereof. In some embodiments, the natural biomaterials include, by way of non-limiting examples, collagen, fibronectin, laminin and other extracellular matrices. In some embodiments, the synthetic biomaterials may include, by way of non-limiting examples, hydroxyapatite, alginate, agarose, poly (glycolic acid), poly (lactic acid) and their copolymers. In some embodiments, a support substrate is solid. In some embodiments, a support substrate is semi-solid. In additional embodiments, a support substrate is a combination of solid and semi-solid support elements.
[0101] In some embodiments, the support substrate is raised or raised above a non-permeable surface, such as a portion of a cell culture environment (e.g., a Petri dish, a cell culture flask, etc.) or a bioreactor In still other embodiments, a raised support substrate further facilitates circulation of the cell culture medium and improves contact with all surfaces of the multicellular bodies and / or layers.
[0102] Methods for making multicellular bodies
[0103] There are several ways to prepare multicellular bodies that have the characteristics described in this document. In some embodiments, a multicellular body can be prepared from a cell paste containing a plurality of living cells or with a desired cell density and viscosity. In additional embodiments, the cell paste can be formed into a desired shape and a multicellular body is formed by maturation (e.g., incubation). In a particular embodiment, a multicellular body is produced by forming a cell paste that includes a plurality of living cells in a desired shape (e.g., a cylinder, a sphere). In additional embodiments, the cell paste is incubated in a controlled environment to allow the cells to adhere and / or cohere with each other to form the multicellular body. In another particular embodiment, a multicellular body is produced by forming a cell paste that includes a plurality of living cells in a device that holds the cell paste in a three dimensional shape. In realizations In addition, the cell paste is incubated in a controlled environment, while remaining in the three-dimensional form for a sufficient time to produce a body having sufficient cohesion to attach itself to a flat surface, as described herein. document.
[0104]
[0105] In various embodiments, a cell paste is provided by: (A) the mixture of cells or aggregates of cells (from one of several cell types) and a cell culture medium (eg, in a predetermined ratio) to result in a suspension of cells, and (B) compacting the cell suspension to produce a cell paste with a desired cell density and viscosity. In various embodiments, compaction is achieved by a number of methods, such as by the concentration of a particular cell suspension that is the result of a cell culture to achieve the desired cell concentration (density), the viscosity and the required consistency for cell paste. In a particular embodiment, a relatively dilute cell suspension from the cell culture is centrifuged for a determined time to reach a concentration of cells in the pellet which allows conformation in a mold. Tangential flow filtration ("TFF") is another suitable method for concentrating or compacting cells. In some embodiments, the compounds are combined with the cell suspension to confer the required extrusion properties. Suitable compounds include, by way of non-limiting examples, collagen, hydrogels, Matrigel, nanofibers, self-assembling nanofibers, gelatin, fibrinogen, etc.
[0106]
[0107] In some embodiments, the cell paste is produced by mixing a plurality of living cells with a tissue culture medium, and compaction of living cells (e.g., by centrifugation). One or more components of the ECM (or derivatives of a component of the ECM) are optionally included by resuspending the cell pellet in one or more physiologically acceptable buffers containing the one or more components of the ECM (or one or more derivatives of the or components of the ECM) and the resulting cell suspension is centrifuged again to form the cell paste.
[0108]
[0109] In some embodiments, the cell density of the cell paste desired for further processing may vary with cell types. In further embodiments, the interactions between the cells determine the properties of cell paste, and different cell types will have a different relationship between cell density and cell-cell interaction. In yet other embodiments, cells can be pretreated to increase cellular interactions prior to cell paste formation. For example, cells can be incubated inside a centrifuge tube after centrifugation, in order to improve cell-cell interactions before the cell paste is formed.
[0110]
[0111] In various embodiments, many methods are used to shape the cell paste. For example, in a particular embodiment, the cell paste is molded or pressed manually (eg, after concentration / compaction) to achieve a desired shape. By way of a further example, the cell paste can be collected (eg, aspirated) in a preformed instrument, such as a micropipette (e.g., a capillary pipette), which forms the cell paste to conform to a surface inside of the instrument. The cross-sectional shape of the micropipette (e.g., a capillary pipette) is alternately circular, square, rectangular, triangular, or other non-circular cross-sectional shape. In some embodiments, the cell paste is formed by depositing in a preformed mold, such as a plastic mold, metal mold or gel mold. In some embodiments, a centrifugal molding or continuous molding is used to shape the cell paste.
[0112]
[0113] Referring to FIG. 5, in one particular example, the shaping includes retaining the cell paste 4 in a shaping device 5 (eg, a capillary pipette) to allow the cells to partially adhere and / or cohere with each other. in the shaping device. By way of further example, cell paste can be aspirated into a shaping device and held in the shaping device for a period of maturation (also referred to herein as an incubation period) to allow at least partially, the cells adhere and / or cohere with each other. In some embodiments, the shaping device (eg, a capillary pipette) is part of a printhead of a bioprinter or similar device, operative to automatically place the multicellular body in a three-dimensional artificial structure. However, there is a limit on the amount of time that cells can remain in a shaping device, such as a capillary pipette, which provides cells with only limited access at best, to oxygen and / or the nutrients, before the viability of the cells is affected.
[0114]
[0115] In some embodiments, a partially adhered and / or cohesive cell paste is transferred from the shaping device (eg, a capillary pipette) to a second shaping device (eg, a mold) that allows nutrients and / or the oxygen is supplied to the cells while they remain in the second shaping device for a further period of maturation. An example of a suitable shaping device that allows nutrients and oxygen to be delivered to the cells is a template for the production of a plurality of multicellular bodies (eg, substantially identical multicellular bodies). By way of further example, such a mold includes a biocompatible substrate based on a material that is resistant to migration and inward growth of cells within the substrate and that is resistant to adhesion of cells to the substrate . In various embodiments, the substrate may be suitably formed of Teflon®, (PTFE), stainless steel, agarose, polyethylene glycol, glass, metal, plastic or gel materials (for example, agarose gel or other hydrogel), and similar materials. In some embodiments, the mold is also suitably configured to allow delivery of a tissue culture medium to the cell paste (eg, by supplying the tissue culture media on top of the mold). In a particular embodiment, a plurality of elongated slots are formed in the substrate. In a further particular embodiment, the depth of each slot is in the range of about 500 microns to about 1000 microns, and the bottom of each slot has a semicircular cross-sectional shape for the formation of elongated cylindrical multicell bodies having a shape with substantially circular cross section. In a particular additional embodiment, the width of the grooves is suitably a little larger than the width of the multicellular body to be produced in the mold. For example, the width of the slots is suitably in the range from about 300 microns to about 1000 microns.
[0116] Therefore, in embodiments where a second shaping device is used, the paste of partially adhered and / or cohesive cells is transferred from the first shaping device (eg, a capillary pipette) to the second shaping device (for example, a capillary pipette). example, a mold). In further embodiments, the paste of partially adhered and / or cohesive cells can be transferred through the first shaping device (e.g., the capillary pipette) to the slots of a mold. In yet other embodiments, after a period of maturation in which the mold is incubated together with the cell paste retained therein, in a controlled environment to allow the cells in the cell paste to adhere and / or cohere additionally to each other to form the multicellular body, the cohesion of the cells will be strong enough to allow the resulting multicellular body to be collected with an instrument (e.g., a capillary pipette). In still other embodiments, the capillary pipette is suitably part of a printhead of a bioprinter or similar apparatus that can act to automatically position the multicellular body within a three dimensional artificial structure.
[0117] In some embodiments, the shape of the cross section and the size of the multicellular bodies will substantially correspond to the shapes of the cross section and the sizes of the first shaping device and, optionally, the second shaping device used to prepare the multicellular bodies. , and the person skilled in the art will be able to select suitable shaping devices having cross-sectional shapes, cross-sectional surfaces, diameters and lengths suitable for the creation of multicellular bodies having the cross-sectional shapes, cross-sectional surfaces , diameters and lengths described above.
[0118] As described herein, a wide variety of cell types may be employed to make the multicellular bodies of the present embodiments. Therefore, one or more cell types or aggregates of cells including, for example, all cell types mentioned herein, can be used as starting materials to create the cell paste. For example, cells, such as non-human myocytes, endothelial cells, fat cells and fibroblasts are optionally employed. As described herein, a multicellular body is homocellular or heterocellular. In order to prepare homocellular multicellular bodies, the cell paste is suitably homocellular, that is, it includes a plurality of living cells of a single cell type. To prepare heterocellular multicellular bodies, on the other hand, the cell paste will conveniently include a significant number of cells of more than one cell type (ie, cell paste will be heterocellular). As described herein, when a pulp of heterocellular cells is used to create the multicellular bodies, the living cells can be "sorted" during the maturation and cohesion process based on differences in the adhesive forces of the cells, and can recover its physiological conformation.
[0119] In some embodiments, in addition to the plurality of living cells, one or more components of the ECM or one or more derivatives of one or more components of the ECM (eg, gelatin, fibrinogen, collagen, fibronectin, laminin, elastin and / or proteoglycans) can be suitably included in the cell paste to incorporate these substances into the multicellular bodies, as has been pointed out herein. In additional embodiments, the addition of ECM components or derivatives of ECM components to the cell paste can promote the cohesion of the cells in the multicellular body. For example, gelatin and / or fibrinogen are optionally added to the cell paste. More particularly, a solution of 10-30% gelatin and a solution of 10-80 mg / ml fibrinogen are optionally mixed with a plurality of living cells to form a cell suspension containing gelatin and fibrinogen.
[0120] Various methods are available to facilitate the additional maturation process. In one embodiment, the cell paste can be incubated at about 37 ° C for a period of time (which may depend on the cell type) to promote adhesion and / or cohesion. Alternatively or in addition, the cell paste may be preserved in the presence of the cell culture medium containing factors and / or ions to promote adhesion and / or cohesion.
[0121] Arrangement of the multicellular bodies on a support substrate to form layers
[0122] A number of methods are suitable for arranging the multicellular bodies on a support substrate for produce a desired three-dimensional structure (e.g., a substantially planar layer). For example, in some embodiments, the multicellular bodies are manually placed in contact with each other, deposited in place by extrusion from a pipette, nozzle or needle or positioned in contact with an automated machine, such as a bioprinter. .
[0123] As described herein, in some embodiments, the support substrate is permeable to fluids, gases and nutrients and allows the cell culture media to come in contact with all surfaces of the multicellular bodies and / or the layers during the arrangement and the subsequent fusion. As further described herein, in some embodiments, a support substrate is made from natural biomaterials such as collagen, fibronectin, laminin and other extracellular matrices. In some embodiments, a support substrate is made of synthetic biomaterials, such as hydroxyapatite, alginate, agarose, poly (glycolic acid), poly (lactic acid) and their copolymers. In some embodiments, a support substrate is solid. In some embodiments, a support substrate is semi-solid. In other embodiments, a support substrate is a combination of solid and semi-solid support elements. In additional embodiments, a support substrate is flat to facilitate the production of planar layers. In some embodiments, the support substrate is raised or raised above a non-permeable surface, such as a portion of a cell culture environment (e.g., a Petri dish, a cell culture flask, etc.) or a bioreactor Therefore, in some embodiments, a high permeable support substrate contributes to the prevention of premature cell death, contributes to the improvement of cell growth and facilitates fusion of the multicellular bodies to form layers.
[0124] As described in this document, in various embodiments, multicellular bodies have many shapes and sizes. In some embodiments, the multicellular bodies are elongated and cylinder-shaped. See, for example, Figs. 1 and 3. In some embodiments, the multicellular bodies provided herein have similar lengths and / or diameters. In other embodiments, the multicellular bodies provided herein have different lengths and / or diameters. In some embodiments, the multicellular bodies are substantially spherical. See, for example, Figs. 2 and 4. In some embodiments, the layers include substantially spherical multicellular bodies that have a substantially similar size. In other embodiments, the layers include substantially spherical multicellular bodies that have different sizes.
[0125] Referring to Fig. 6, in some embodiments, the multicellular bodies 1 are disposed on a support substrate 3 horizontally adjacent to, and in contact with, one or more other multicellular bodies to form a substantially planar layer.
[0126] Referring to FIG. 7, in some embodiments, the substantially spherical multicellular bodies 2 are disposed on a support substrate 3 horizontally adjacent to, and in contact with, one or more other substantially spherical multicellular bodies. In further embodiments, this method is repeated to construct a pattern of substantially spherical multicellular bodies, such as a grid, to form a substantially planar layer.
[0127] Referring to Fig. 8, in a particular embodiment, a multicellular body 6 is superimposed on a support substrate 3 through an instrument such as a capillary pipette 5, so that it is adjacent horizontally, and is in contact with one or several other multicellular bodies. In additional embodiments, a multicellular body is placed on a support substrate so that it is parallel to a plurality of other multicellular bodies.
[0128] Referring to FIG. 9, in some embodiments, a subsequent series of multicell bodies 8 are arranged vertically adjacent to, and in contact with, a prior series of multicellular bodies 9 on a support substrate 3 to form a further layer. gross.
[0129] In other embodiments, the layers of different shapes and sizes are formed by the arrangement of multicellular bodies of various shapes and sizes. In some embodiments, the multicellular bodies of various shapes, sizes, densities, cell compositions and / or additive compositions are combined in one layer and contribute, for example, to the appearance, taste and texture of the resulting layer.
[0130] Referring to Fig. 10, in some embodiments, the elongated multicell bodies 9 are arranged adjacently and in contact with substantially spherical multicellular bodies 10 on a support substrate 3 to form a complex layer.
[0131] Once the assembly of a layer is complete, in some embodiments, a tissue culture medium is poured onto the upper part of the artificial structure. In additional embodiments, the tissue culture medium enters the spaces between the multicellular bodies to support the cells in the multicellular bodies. The multicellular bodies in the three-dimensional artificial structure can be fused together to produce a substantially flat layer for use in the formation of artificially produced edible meat. By "fusing", "fusing" or "fusing", it is understood that the cells of the contiguous multicellular bodies adhere and / or cohere with each other, either directly through interactions between the cell surface proteins or indirectly through interactions of cells with extracellular matrix (ECM) components or derived from ECM components. In some embodiments, the cells within the multicellular bodies produce their own cell-specific ECM (eg, collagen), which provides the mechanical integrity of the multicellular bodies and the edible meat product. In some embodiments, a fused layer is completely fused and the multicell bodies have become substantially contiguous. In some embodiments, a fused layer is substantially fused or partially fused and the cells of the multicellular bodies are adhered and / or cohesive to the extent necessary to allow movement and manipulation of the intact layer.
[0132] In some embodiments, the multicellular bodies are fused to form a layer in a cell culture environment (e.g., a Petri dish, a cell culture flask, a bioreactor, etc.). In additional embodiments, the multicellular bodies are fused to form a layer in an environment with conditions suitable for facilitating the growth of the cell types included in the multicellular bodies. In various embodiments, the fusion takes place for approximately more than 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 minutes, and increases therein. In various other embodiments, fusion occurs for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28 , 30, 32, 34, 36, 38, 40, 42, 44, 46, and 48 hours, and increases therein. In yet other diverse embodiments, fusion takes place for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, and 14 days, and increases therein. In further embodiments, the fusion is carried out for from about 2 hours to about 36 hours. Various factors influence the required fusion time, including, by way of non-limiting examples, cell types, cell type ratios, culture conditions and the presence of additives, such as growth factors.
[0133] Once the fusion of one layer has been completed, in some embodiments, the layer and the support substrate are separated. In other embodiments, the support layer and substrate are separated when the fusion of a layer is substantially complete or partially complete, but the cells of the layer adhere and / or cohere to each other to the extent necessary to allow movement, manipulation and stack the layer without breaking it. In additional embodiments, the support layer and substrate are separated through conventional methods for melting, dissolving or degrading the support substrate. In still other embodiments, the support substrate is dissolved, for example, by a change in temperature, light or other stimuli that do not adversely affect the layer. In a particular embodiment, the support substrate is prepared from a flexible material and peeled from the layer.
[0134] In some embodiments, the separated layer is transferred to a bioreactor for further maturation. In some embodiments, the separated layer matures and is further fused after incorporation into an artificially produced meat product.
[0135] In other embodiments, the support layer and substrate are not separated. In additional embodiments, the support substrate degrades or biodegrades prior to packaging, freezing, selling or consuming the artificially produced meat product assembled.
[0136] Arrangement of the layers on a support substrate to make artificially produced meat
[0137] A number of methods are suitable for arranging the layers on a support substrate to produce artificially produced meat. For example, in some embodiments, the layers are manually placed in contact with each other, or deposited in place by means of an automated, computer-assisted machine such as a bioprinter, according to a computer command sequence. In additional embodiments, the substantially flat layers are stacked to make artificially produced meat.
[0138] In various embodiments, approximately 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or 500 layers or increments thereof are stacked thereon . In further embodiments, the stacking is repeated to develop a thickness that approximates a traditional meat product such as a carpaccio, a bacon strip, a hamburger, a fish fillet, a chicken breast or a steak. In various embodiments, the stacked layers comprise an artificially produced meat product with a thickness of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mm or increments therein.
[0139] In some embodiments, a layer has an orientation defined by the placement, pattern, or orientation of the multicellular bodies. In other embodiments, each layer is stacked with a particular orientation relative to the support substrate and / or one or more other layers. In various embodiments, one or more layers are stacked with an orientation that includes a rotation relative to the support substrate and / or the bottom layer, wherein the rotation is about 5, 10, 15, 20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155 , 160, 165, 170, 175 and 180 degrees or increases in them. In other embodiments, all the layers are oriented substantially in a similar manner.
[0140] Referring to Fig. 11, in a particular embodiment, the layers have an orientation defined by the parallel placement of multicellular bodies used to form the layer. In a further particular embodiment, the layers are stacked with an orientation that includes 90 degrees of rotation with respect to the bottom layer to form artificially produced flesh.
[0141] Once the stacking of the layers is completed, in some embodiments, to the layers in the artificial structure three-dimensional are allowed to merge with each other to produce artificially produced meat. In some embodiments, the layers are fused to form flesh artificially produced in a cell culture environment (eg, a Petri dish, a cell culture flask, a bioreactor, etc.). In various embodiments, the fusion takes place for more than about 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 minutes, and increases therein. In various other embodiments, fusion occurs for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28 , 30, 32, 34, 36, 38, 40, 42, 44, 46 and 48 hours, and increases thereof. In further embodiments, the fusion is carried out for from about 2 hours to about 36 hours.
[0142] In some embodiments, once stacked, the cells of the multicellular bodies and the layers begin to die due to the inability to diffuse gases, liquids and nutrients, or to otherwise reach the interior portions of the artificial structure. In additional embodiments, the gradual death of the cells is similar to the natural cell death that occurs in the tissues of an organism after death. In some embodiments, the layers of the artificially produced artificial meat structure fuse with each other simultaneously with a progressive death of the cells. In some embodiments, the multicellular bodies of the layers continue to fuse with each other simultaneously with the progressive death of the cells. In further embodiments, the fusion within and between the layers is complete or substantially complete before the death of a majority of the cells of the artificial structure. In further embodiments, the fusion within and between the layers is completed or substantially completed before the death of all cells of the artificial structure.
[0143] Once the assembly of the artificially produced meat is completed, in some embodiments, the meat and the support substrate are separated. In additional embodiments, the meat and the support substrate are separated through conventional methods for melting, dissolving or degrading the support substrate. In still other embodiments, the support substrate is dissolved, for example, by a change in temperature, light or other stimuli that do not adversely affect the meat. In a particular embodiment, the support substrate is made of a flexible material and is detached from the meat. In some embodiments, the separated meat is transferred to a bioreactor for further maturation. In other embodiments, the meat and the support substrate are not separated. In additional embodiments, the support substrate degrades or biodegrades prior to sale or consumption.
[0144] In some embodiments, the meat is radiated. In some embodiments, the meat is frozen to prevent decomposition or degradation prior to distribution, sale and consumption. In additional embodiments, the frozen meat is vacuum packed.
[0145] Meat artificially produced
[0146] In the present document, in some embodiments, artificially produced meat products are described, obtained according to the methods of the invention.
[0147] In some embodiments, the artificially produced meat products are fresh. In other embodiments, artificially produced meat products are preserved. In further embodiments, the meat is preserved, for example, by cooking, drying, smoking, canning, pickling, salt curing or freezing.
[0148] In some embodiments, the artificially produced meat products are substantially free of pathogenic microorganisms. In further embodiments, controlled and substantially sterile methods for the preparation of cells, cell culture, preparation of multicellular bodies, preparation of layers and preparation of artificially produced meat give rise to a product substantially free of pathogenic microorganisms. In additional embodiments, an additional advantage of such a product is increased utility and safety.
[0149] In some embodiments, the artificially produced meat products are shaped. In additional embodiments, the meat is formed, for example, by controlling the amount, size and arrangement of the multicellular bodies and / or the layers used for the construction of the meat. In other embodiments, the meat is formed, for example, by cutting, pressing, molding or stamping. In some embodiments, the shape of a meat product is selected to resemble a traditional meat product, such as a strip of bacon, a string of sausages, a Russian steak, a hamburger, a hot dog, a fish fillet, a chicken breast, a chicken strip, pieces of breaded chicken, a meatloaf or a steak. In other embodiments, artificially produced meat products are chopped.
[0150] Examples
[0151] The following illustrative examples are representative of embodiments described herein and are not intended to be limiting in any way.
[0152] Example 1 - Preparation of the support substrate
[0153] To prepare a 2% agarose solution, 2 g of ultrapure low melting point agarose (LMP) was dissolved. in 100 ml of ultrapure water / buffer solution (1: 1, v / v). The buffer solution is optionally PBS (Dulbecco's phosphate-buffered saline 1x) or HBSS (balanced salt solution of Hanks 1x). The agarose solution was placed in a beaker containing hot water (more than 80 ° C) and kept on the hot plate until the agarose completely dissolved. The agarose solution remained liquid, as long as the temperature was above 36 ° C. Below 36 ° C, a phase transition occurs, the viscosity increases and, finally, the agarose forms a gel.
[0154] To prepare the agarose support substrate, 10 ml of 2% liquid agarose (temperature> 40 ° C) was placed in a Petri dish with a diameter of 10 cm and spread horizontally to form a uniform layer. The agarose was allowed to form a gel at 4 ° C in a refrigerator.
[0155] Example 2 - Culture of porcine aortic smooth muscle cells
[0156] Freshly isolated porcine aortic smooth muscle cells (PASMCs) were cultured in low glucose DMEM with 10% fetal bovine serum (Hyclone Laboratories, UT), 10% porcine serum (Invitrogen), L-ascorbic acid, copper sulfate, HEPES, L-proline, L-alanine, L-glycine and penicillin G (all the supplements mentioned above were purchased from Sigma, St. Louis, MO). The cell lines were grown in plates coated with 0.5% gelatin (swine skin gelatin: Sigma) (Techno Plastic Products, St. Louis, MO) and kept at 37 ° C in a humidified atmosphere containing 5% CO2 The PASMCs were subcultured until passage 7, before being used to form the multicellular bodies.
[0157] Example 3 - Preparation of spheroids and multicell cylinders
[0158] The cell cultures were washed twice with phosphate buffered saline (PBS, Invitrogen) and treated for 10 min with 0.1% trypsin (Invitrogen) and centrifuged at 1500 rpm for 5 min. Cells were resuspended in 4 ml of cell-type-specific media and incubated in 10 ml tissue culture flasks (Bellco Glass, Vineland, NJ) at 37 ° C with 5% CO2 on a rotary shaker (New Brunswick Scientific, Edison, NJ) for one hour, to recover adhesion and centrifuged at 3500 rpm. The resulting pellets were transferred to capillary micropipettes of 300 μm (Sutter Instrument, CA) or 500 μm (Drummond Scientific Company, Broomall, PA) diameter and incubated at 37 ° C with 5% CO2 for 15 min. For the spherical multicellular bodies, extruded cylinders were cut into equal fragments that were rounded overnight in a rotary shaker. Depending on the diameter of the micropipettes, this procedure provided regular spheroids with a defined size and number of cells. For the cylindrical multicellular bodies, the cylinders were mechanically extruded in non-adhesive or agarose Teflon® molds, prepared specifically using a bioprinter. After maturing overnight in the mold, the cell cylinders were sufficiently cohesive to be deposited.
[0159] The multicellular bodies were packed in cartridges (micropipettes of 300-500 μm internal diameter). The cartridges were inserted into a bioprinter and distributed on a support substrate in accordance with a sequence of computer commands, which encoded the shape of the structure to be printed.
[0160] Example 4 - Preparation of artificially produced meat
[0161] The multicellular cylindrical bodies are prepared as described in Example 3. The multicellular bodies are heterocellular and are composed of the PASMCs of Example 2 and porcine coronary artery endothelial cells (PCAEC, Genlantis, San Diego, CA, Product No. PP30005 ). The relationship between myocytes and endothelial cells in multicellular bodies is approximately 6: 1. The multicellular bodies have a cross-sectional diameter of 300 μm and a length of 2 cm, 3 cm, 4 cm or 5 cm. The multicellular and matured bodies are packed in cartridges (micropipettes of 300 μm internal diameter), which are then inserted into a bioprinter.
[0162] An agarose support substrate is prepared as described in Example 1. The support substrate is raised above the bottom of a large Petri dish by a fine mesh pedestal, such that the culture medium of the cells can be brought into contact with all the surfaces of the multicellular bodies and the layers deposited on the substrate.
[0163] A bioprinter distributes the multicellular bodies on the support substrate according to the instructions of a computer command sequence. The script encodes the placement of the cylindrical multicellular bodies to form a substantially square monolayer with an average width of approximately 10 cm and an average length of approximately 10 cm. The multicellular bodies are placed parallel to each other with bodies of different lengths, placed end to end to form the coded form.
[0164] The culture medium is poured over the top of the layer and the artificial structure is allowed to partially fuse during the course of about 12 hours at 37 ° C in a humidified atmosphere containing 5% CO2. During this time, the cells of the multicellular bodies adhere and / or cohere to the extent necessary to allow movement and manipulation of the layer without breaking it.
[0165] The partially fused layers are separated from the support and stacked. Sixty-five layers are stacked to form the artificially produced meat, which has a total width and height of approximately 2 cm and a length and width of approximately 10 cm. Each layer is rotated 90 degrees with respect to the layer below. Once stacked, the cells begin to die due to lack of oxygen, since the culture medium is not changed. Cell death begins inside the stack, since these cells are the first to be deprived of oxygen, and progressively reach the outer cells, since oxygen is gradually depleted in the surrounding culture medium. Simultaneously with cell death, the partially fused layers continue to merge, although they also begin to fuse in a vertical direction. Since the fusion process lasts about 6 hours, although cell death lasts around 20 hours, the artificial structure after cell death is fully fused and assumes a shape similar to a square pork burger.

权利要求:
Claims (8)
[1]
1. A method for making artificially produced meat, the method comprising:
preparing a plurality of multicellular bodies comprising a plurality of non-human myocytes coalesced with each other;
superimposing more than one multicellular body adjacently on a flat support substrate; fusing said multicellular bodies at least partially to each other to form a first layer; stack more than 50 additional layers on the first layer;
merge the stacked layers to form a volume of artificially produced meat; Y
cultivate the stacked layers to fuse the layers while the layers in an inner region of the volume die so that most of the cells in the volume are dead after completing the fusion between the layers, at least partially, and
where artificially produced meat is edible.
[2]
The method according to claim 1, further comprising freezing said volume of artificially produced meat.
[3]
The method according to claim 1, wherein the preparation of the plurality of multicellular bodies comprises the preparation of a plurality of elongated multicellular bodies comprising a plurality of non-human myocytes coalesced with each other and the preparation of a plurality of multicellular bodies substantially spherical comprising a plurality of non-human myocytes coalesced with each other; and further wherein the overlap of more than one multicellular body comprises overlaying more than one elongated multicellular body and more than one substantially spherical multicellular body adjacently on a flat support substrate.
[4]
The method according to claim 3, wherein said elongated multicell bodies have a length ranging from 1 mm to 10 cm.
[5]
The method according to claim 1, wherein the preparation of the plurality of multicellular bodies comprises the preparation of a plurality of substantially spherical multicellular bodies comprising a plurality of non-human myocytes coalesced with each other; and further wherein the overlap of more than one multicellular body comprises superimposing more than one substantially spherical multicellular body adjacently on a flat support substrate.
[6]
The method according to claim 1, wherein the flat support substrate is permeable to liquids and nutrients and allows the cell culture medium to contact all the surfaces of the multicellular bodies.
[7]
The method according to claim 1, wherein said multicellular bodies have a diameter of 50 jm to 1000 | jm to allow a diffusion in order to sufficiently support the maintenance and growth of said non-human myocytes and endothelial cells non-human in culture.
[8]
The method according to claim 1, wherein said multicellular bodies have a diameter of 100 jm to 500 jm.

类似技术:

---

公开号 | 公开日 | 专利标题

---

ES2685638T3|2018-10-10|Artificially produced edible meat

---

EP2831291B1|2019-04-24|Engineered leather and methods of manufacture thereof

---

KR20160043146A|2016-04-20|Self-assembling multicellular bodies and methods of producing a three-dimensional biological structure using the same

---

CN109153182A|2019-01-04|For the method to 3 D-printing supply ink and use the 3 D-printing method of this method

---

EA018218B1|2013-06-28|Method for producing non-human tissue engineered meat products in vitro and products produced by said method

---

CN103534346A|2014-01-22|Cell-synthesized particles

---

US20110301249A1|2011-12-08|Stem cell enhanced protein products and uses therof

---

Bomkamp et al.2022|Scaffolding biomaterials for 3D cultivated meat: Prospects and challenges

---

KR20220012204A|2022-02-03|Method for manufacturing cultured meat based on cell coating and cultured meat prepared therefrom

---

JP2021534780A|2021-12-16|Equipment and methods for producing tissue from cells

---

KR20220030199A|2022-03-10|Porous cell support containing plant protein and cultured meat prepared using the same

---

US20210348129A1|2021-11-11|Non-decellularized plant leaf cultures for meat

---

CN101132803A|2008-02-27|Tissue engineering edible meat and method of manufacturing the same

---

Raman2021|Biofabrication

---

AU2020322793A1|2022-03-03|Scalable bioreactor systems and methods for tissue engineering

---

WO2021021968A1|2021-02-04|Scalable bioreactor systems and methods for tissue engineering

同族专利:

---

公开号 | 公开日

---

PL2736357T3|2019-02-28|

---

CN103747693A|2014-04-23|

---

ES2685638T3|2018-10-10|

---

CN103747693B|2017-08-01|

---

AU2018214029A1|2018-08-23|

---

US20140093618A1|2014-04-03|

---

EP2736357B9|2019-01-09|

---

US20130029008A1|2013-01-31|

---

EP2736357B1|2018-05-02|

---

AU2016204474A1|2016-07-21|

---

AU2016204474B2|2018-08-23|

---

WO2013016547A2|2013-01-31|

---

KR20140050064A|2014-04-28|

---

PL2736357T4|2019-02-28|

---

EP2736357A4|2015-05-06|

---

US8703216B2|2014-04-22|

---

EP2736357A2|2014-06-04|

---

AU2012286817A1|2014-02-13|

---

JP2014521336A|2014-08-28|

---

WO2013016547A3|2013-05-10|

---

CA2842837A1|2013-01-31|

---

JP6523683B2|2019-06-05|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US4291992A|1979-05-22|1981-09-29|R. C. Sanders Technology Systems, Inc.|Printer pin control circuitry|

---

US4247279A|1980-04-18|1981-01-27|Masters William E|Rotational molding system|

---

US4646106A|1982-01-04|1987-02-24|Exxon Printing Systems, Inc.|Method of operating an ink jet|

---

US4772141A|1982-10-27|1988-09-20|Royden C. Sanders, Jr.|Dot matrix printhead pin driver and method of assembly|

---

US4665492A|1984-07-02|1987-05-12|Masters William E|Computer automated manufacturing process and system|

---

US4585139A|1985-08-08|1986-04-29|Sanders Associates, Inc.|Cooperating cover mechanisms|

---

US4673304A|1985-08-13|1987-06-16|Sanders Associates, Inc.|Thermal printer ribbon cartridge for wide ribbons|

---

US4594597A|1985-08-13|1986-06-10|Sanders Associates, Inc.|Thermal printer|

---

DE3728155A1|1986-12-03|1987-12-10|Inst Hochseefischerei|METHOD AND INSTALLATION FOR PRODUCING STRUCTURED PRODUCTS|

---

US5039297A|1987-07-28|1991-08-13|Masters William E|Rotational molding apparatus|

---

US5075805A|1988-02-25|1991-12-24|Tandon Corporation|Disk drive controller system|

---

US5016121A|1988-02-25|1991-05-14|Tandon Corporation|Disk drive controller system|

---

US4969758A|1988-03-24|1990-11-13|Royden C. Sanders, Jr.|Method of improving the printing speed of an impact dot printer printing in a near letter quality mode|

---

US4980112A|1988-06-24|1990-12-25|Masters William E|Method for rotational molding|

---

US5040911A|1988-08-10|1991-08-20|Royden C. Sanders, Jr.|Paper advancing system for high speed printers|

---

US4896980A|1988-08-10|1990-01-30|Royden C. Sanders, Jr.|Paper advancing system for high speed printers|

---

US4921365A|1988-08-10|1990-05-01|Royden C. Sanders, Jr.|High speed shuttle printer|

---

US4889438A|1989-04-12|1989-12-26|Royden C. Sanders, Jr.|Serial printer carriage drive with ballistic rebound reversal|

---

US4948280A|1989-04-12|1990-08-14|Royden C. Sanders, Jr.|Serial printer carriage drive with ballistic rebound reversal|

---

US5216616A|1989-06-26|1993-06-01|Masters William E|System and method for computer automated manufacture with reduced object shape distortion|

---

US5134569A|1989-06-26|1992-07-28|Masters William E|System and method for computer automated manufacturing using fluent material|

---

US5204055A|1989-12-08|1993-04-20|Massachusetts Institute Of Technology|Three-dimensional printing techniques|

---

SE9003712L|1990-11-22|1992-01-07|Kabi Pharmacia Ab|GEL-PHARMACEUTICAL LIQUID COMPOSITION AND APPLICATION THEREOF IN PHARMACEUTICAL COMPOSITIONS|

---

US5697324A|1993-06-25|1997-12-16|Van Der Lely; Cornelis|Apparatus for automatically milking animals, such as cows|

---

US5490962A|1993-10-18|1996-02-13|Massachusetts Institute Of Technology|Preparation of medical devices by solid free-form fabrication methods|

---

US5605662A|1993-11-01|1997-02-25|Nanogen, Inc.|Active programmable electronic devices for molecular biological analysis and diagnostics|

---

US5546313A|1994-09-12|1996-08-13|Masters; William E.|Method and apparatus for producing three-dimensional articles from a computer generated design|

---

CH688960A5|1994-11-24|1998-06-30|Pelikan Produktions Ag|Droplet generator for microdroplets, especially for an inkjet printer.|

---

US6239273B1|1995-02-27|2001-05-29|Affymetrix, Inc.|Printing molecular library arrays|

---

US5599695A|1995-02-27|1997-02-04|Affymetrix, Inc.|Printing molecular library arrays using deprotection agents solely in the vapor phase|

---

JPH08254446A|1995-03-16|1996-10-01|Fujitsu Ltd|Ultrasonic printing method and device as well as formation of acoustic lens|

---

US5658802A|1995-09-07|1997-08-19|Microfab Technologies, Inc.|Method and apparatus for making miniaturized diagnostic arrays|

---

US5702717A|1995-10-25|1997-12-30|Macromed, Inc.|Thermosensitive biodegradable polymers based on polyblock copolymers|

---

US6260938B1|1996-04-23|2001-07-17|Canon Kabushiki Kaisha|Ink-jet printing method and apparatus for printing with inks of different densities|

---

US6387707B1|1996-04-25|2002-05-14|Bioarray Solutions|Array Cytometry|

---

US6548263B1|1997-05-29|2003-04-15|Cellomics, Inc.|Miniaturized cell array methods and apparatus for cell-based screening|

---

GB9611582D0|1996-06-04|1996-08-07|Thin Film Technology Consultan|3D printing and forming of structures|

---

US5797898A|1996-07-02|1998-08-25|Massachusetts Institute Of Technology|Microchip drug delivery devices|

---

WO1998030395A1|1997-01-08|1998-07-16|Kabushiki Kaisha Tec|Ink jet printer|

---

US6280771B1|1997-02-20|2001-08-28|Therics, Inc.|Dosage forms exhibiting multi-phasic release kinetics and methods of manufacture thereof|

---

AU6868598A|1997-03-20|1998-10-12|Therics, Inc.|Fabrication of tissue products using a mold formed by solid free-form methods|

---

US6341952B2|1997-03-20|2002-01-29|Therics, Inc.|Fabrication of tissue products with additives by casting or molding using a mold formed by solid free-form methods|

---

US6213168B1|1997-03-31|2001-04-10|Therics, Inc.|Apparatus and method for dispensing of powders|

---

US6109717A|1997-05-13|2000-08-29|Sarnoff Corporation|Multi-element fluid delivery apparatus and methods|

---

EP1019492A1|1997-07-03|2000-07-19|Massachusetts Institute Of Technology|Tissue-engineered constructs|

---

WO1999031222A1|1997-12-18|1999-06-24|Willem Frederik Van Eelen|Industrial scale production of meat from in vitro cell cultures|

---

US6087102A|1998-01-07|2000-07-11|Clontech Laboratories, Inc.|Polymeric arrays and methods for their use in binding assays|

---

US6419883B1|1998-01-16|2002-07-16|University Of Washington|Chemical synthesis using solvent microdroplets|

---

US6197575B1|1998-03-18|2001-03-06|Massachusetts Institute Of Technology|Vascularized perfused microtissue/micro-organ arrays|

---

US6103528A|1998-04-17|2000-08-15|Battelle Memorial Institute|Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices|

---

US6139831A|1998-05-28|2000-10-31|The Rockfeller University|Apparatus and method for immobilizing molecules onto a substrate|

---

US6132468A|1998-09-10|2000-10-17|Mansmann; Kevin A.|Arthroscopic replacement of cartilage using flexible inflatable envelopes|

---

WO2000021470A1|1998-10-12|2000-04-20|Therics, Inc.|Composites for tissue regeneration and methods of manufacture thereof|

---

US6547994B1|1998-11-13|2003-04-15|Therics, Inc.|Rapid prototyping and manufacturing process|

---

US6451346B1|1998-12-23|2002-09-17|Amgen Inc|Biodegradable pH/thermosensitive hydrogels for sustained delivery of biologically active agents|

---

JP2000190480A|1998-12-25|2000-07-11|Canon Inc|Ink print method and ink-printing apparatus|

---

SE9900378D0|1999-02-05|1999-02-05|Forskarpatent I Syd Ab|Gels with shape memory|

---

US6979670B1|1999-03-10|2005-12-27|Biora Bioex Ab|Matrix protein compositions for grafting|

---

AT288478T|1999-04-30|2005-02-15|Massachusetts Gen Hospital|PREPARATION OF THREE-DIMENSIONAL VASCULARIZED TISSUE BY USING TWO-DIMENSIONAL MICRO-MADE SHAPES|

---

US6171797B1|1999-10-20|2001-01-09|Agilent Technologies Inc.|Methods of making polymeric arrays|

---

US6503273B1|1999-11-22|2003-01-07|Cyograft Tissue Engineering, Inc.|Tissue engineered blood vessels and methods and apparatus for their manufacture|

---

US6497510B1|1999-12-22|2002-12-24|Eastman Kodak Company|Deflection enhancement for continuous ink jet printers|

---

AU3119601A|2000-01-27|2001-08-07|Gen Hospital Corp|Delivery of therapeutic biological from implantable tissue matrices|

---

US7163712B2|2000-03-03|2007-01-16|Duke University|Microstamping activated polymer surfaces|

---

DE10013223C2|2000-03-13|2002-07-18|Co Don Ag|Process for the in vitro production of three-dimensional, vital cartilage or bone tissue and its use as a transplant material|

---

US6368558B1|2000-03-21|2002-04-09|The Board Of Trustees Of The University Of Illinois|Colorimetric artificial nose having an array of dyes and method for artificial olfaction|

---

US6772026B2|2000-04-05|2004-08-03|Therics, Inc.|System and method for rapidly customizing design, manufacture and/or selection of biomedical devices|

---

DE10018987A1|2000-04-17|2001-10-31|Envision Technologies Gmbh|Device and method for producing three-dimensional objects|

---

US6536873B1|2000-06-30|2003-03-25|Eastman Kodak Company|Drop-on-demand ink jet printer capable of directional control of ink drop ejection and method of assembling the printer|

---

EP1301177A2|2000-07-10|2003-04-16|Therics, Inc.|Method and materials for controlling migration of binder liquid in a powder|

---

US20020182633A1|2000-07-11|2002-12-05|Chen Christopher S.|Methods of patterning protein and cell adhesivity|

---

US6841617B2|2000-09-28|2005-01-11|Battelle Memorial Institute|Thermogelling biodegradable aqueous polymer solution|

---

US20020089561A1|2000-11-09|2002-07-11|Therics, Inc.|Method and apparatus for obtaining information about a dispensed fluid, such as using optical fiber to obtain diagnostic information about a fluid at a printhead during printing|

---

WO2002038280A2|2000-11-10|2002-05-16|Therics, Inc.|A wetting-resistant nozzle for dispensing small volumes of liquid and a method for manufacturing a wetting-resistant nozzle|

---

US6835390B1|2000-11-17|2004-12-28|Jon Vein|Method for producing tissue engineered meat for consumption|

---

US6849423B2|2000-11-29|2005-02-01|Picoliter Inc|Focused acoustics for detection and sorting of fluid volumes|

---

US20020064809A1|2000-11-29|2002-05-30|Mutz Mitchell W.|Focused acoustic ejection cell sorting system and method|

---

US20020064808A1|2000-11-29|2002-05-30|Mutz Mitchell W.|Focused acoustic energy for ejecting cells from a fluid|

---

EP1404516A2|2000-12-13|2004-04-07|Purdue Research Foundation|Microencapsulation of drugs by solvent exchange|

---

US6394585B1|2000-12-15|2002-05-28|Eastman Kodak Company|Ink jet printing using drop-on-demand techniques for continuous tone printing|

---

US6527378B2|2001-04-20|2003-03-04|Hewlett-Packard Company|Thermal ink jet defect tolerant resistor design|

---

US6565176B2|2001-05-25|2003-05-20|Lexmark International, Inc.|Long-life stable-jetting thermal ink jet printer|

---

AU2002365110A1|2001-07-10|2003-07-15|Massachusetts Institute Of Technology|Small molecule microarrays|

---

US6986739B2|2001-08-23|2006-01-17|Sciperio, Inc.|Architecture tool and methods of use|

---

US6561642B2|2001-09-28|2003-05-13|Hewlett-Packard Development Company|Ink jet printer system for printing an image on a web overlaying a removable substrate and method of assembling the printer system|

---

KR100438709B1|2001-12-18|2004-07-05|삼성전자주식회사|Ink jet print head|

---

AU2003224710A1|2002-03-18|2003-10-08|Carnegie Mellon University|Method and apparatus for preparing biomimetic scaffold|

---

US7641898B2|2003-03-21|2010-01-05|Materials Evolution And Development Usa, Inc.|Keratinocyte-fibrocyte concomitant grafting for wound healing|

---

US7051654B2|2003-05-30|2006-05-30|Clemson University|Ink-jet printing of viable cells|

---

US20050276791A1|2004-02-20|2005-12-15|The Ohio State University|Multi-layer polymer scaffolds|

---

WO2005081970A2|2004-02-24|2005-09-09|The Curators Of The University Of Missouri|Self-assembling cell aggregates and methods of making engineered tissue using the same|

---

US20090142307A1|2004-07-09|2009-06-04|Athanasiou Kyriacos A|Shape-Based Approach for Scaffoldless Tissue Engineering|

---

US7939003B2|2004-08-11|2011-05-10|Cornell Research Foundation, Inc.|Modular fabrication systems and methods|

---

US20060121006A1|2004-09-10|2006-06-08|Chancellor Michael B|Production of nutritional and therapeutic products from cultured animal cells|

---

CN101132803A|2004-09-17|2008-02-27|乔恩·维恩|Tissue engineering edible meat and method of manufacturing the same|

---

US8690957B2|2005-12-21|2014-04-08|Warsaw Orthopedic, Inc.|Bone graft composition, method and implant|

---

US20070231787A1|2006-04-04|2007-10-04|Voelker Mark A|Methods and devices for imaging and manipulating biological samples|

---

WO2007124023A2|2006-04-21|2007-11-01|Wake Forest University Health Sciences|Ink-jet printing of tissues|

---

EP2090584A1|2008-02-13|2009-08-19|Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V.|Identification of a novel cysteine-rich cell penetrating peptide|

---

US20090248145A1|2008-03-28|2009-10-01|Nanyang Technological University|Method of forming a three-dimensional structure of unidirectionally aligned cells|

---

CA2949615A1|2008-06-24|2010-01-21|The Curators Of The University Of Missouri|Self-assembling multicellular bodies and methods of producing a three-dimensional biological structure using the same|

---

WO2010030964A2|2008-09-12|2010-03-18|The Brigham And Women's Hospital, Inc.|3-dimensional multi-layered hydrogels and methods of making the same|CN103350572B|2013-07-18|2016-05-25|符晓友|The stacking Method of printing of 3D and the stacking printer of 3D|

---

WO2015038988A1|2013-09-13|2015-03-19|Modern Meadow, Inc.|Edible and animal-product-free microcarriers for engineered meat|

---

JP6728049B2|2013-10-30|2020-07-22|ザ キュレイターズ オブ ザ ユニバーシティ オブ ミズーリ|Expandable skeletal muscle lineage formation and culture methods|

---

CA2938156A1|2014-02-05|2015-08-13|Modern Meadow, Inc.|Dried food products formed from cultured muscle cells|

---

AU2016290223B2|2015-07-09|2020-10-08|Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd|3-dimensional printing of food|

---

EP3362266B1|2015-10-15|2021-09-01|The Regents of the University of California|Apparatus and method for cryogenic 3d printing|

---

EP3747901A1|2016-02-15|2020-12-09|Modern Meadow, Inc.|Biofabricated material containing collagen fibrils|

---

WO2018011805A2|2016-07-11|2018-01-18|Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.|Systems and methods for growing cells in vitro|

---

CN106282093A|2016-10-08|2017-01-04|上海生乐康生物技术发展有限公司|A kind of production method of edible cell|

---

US20200100525A1|2017-04-09|2020-04-02|Supermeat The Essence Of Meat Ltd.|Cultured meat-containing hybrid food|

---

AU2018253595A1|2017-11-13|2019-05-30|Modern Meadow, Inc.|Biofabricated leather articles having zonal properties|

---

US11266085B2|2017-11-14|2022-03-08|Ecovative Design Llc|Increased homogeneity of mycological biopolymer grown into void space|

---

GB2573327B|2018-05-03|2021-05-12|Mosa Meat Bv|Apparatus and process for production of tissue from cells|

---

US10669524B2|2018-06-12|2020-06-02|Fork & Goode, Inc.|Large scale cell culture system for making meat and associated products|

---

KR20210052438A|2018-08-07|2021-05-10|노바미트 테크, 에스.엘.|Method for producing edible microextruded product containing protein, composition obtained thereby, and use thereof|

---

CN113038841A|2018-11-15|2021-06-25|阿利夫农场公司|High-quality cultured meat, composition, and method for producing high-quality cultured meat, composition|

---

WO2020131661A1|2018-12-21|2020-06-25|Trustees of Tuffs College|In vitro insect muscle as a nutrition source|

---

WO2021007359A1|2019-07-08|2021-01-14|Meatech 3D, Ltd.|Cultured edible meat fabrication using bio-printing|

---

US11147300B2|2019-11-20|2021-10-19|Upside Foods, Inc.|Apparatuses and systems for preparing a meat product|

---

US20210235733A1|2020-02-04|2021-08-05|Memphis Meats, Inc.|Characteristics of meat products|

---

WO2021250291A1|2020-06-12|2021-12-16|Biotech Foods S.L.|Edible and sterilizable porous 3d scaffold and uses thereof|

---

WO2022019686A1|2020-07-22|2022-01-27|연세대학교 산학협력단|Method for preparing cultured meat on basis of cell coating technique, and cultured meat prepared thereby|

法律状态:

优先权:

---

申请号 | 申请日 | 专利标题

---

US201161511948P| true| 2011-07-26|2011-07-26|

---

US201161511948P|2011-07-26|

---

PCT/US2012/048357|WO2013016547A2|2011-07-26|2012-07-26|Engineered comestible meat|

---