Bioreactor-based processing system for clean meat production

Abstract

The present invention relates to a closed environment process for growing and differentiating cells and culturing cells to confluence to produce tissue, which may be a clean meat product.

Description

[Technical field]

[0001] Efficient, closed continuous, semi-continuous or batch cell and tissue culture systems are needed, for example, for the production of cells, clean meat or other tissues. Current systems are cumbersome to use, expensive to operate, and / or not suitable for scale-up to provide industrial-scale operations. One such example of a prior art device is described in U.S. Pat. No. 8,492,140 (the '140 patent). The device of the '140 patent is a bench-top, laboratory-scale device specifically designed to generate autologous tissue grafts for patients, and is not suitable for industrial-scale product generation, nor is it suitable for scale-up to an industrial-scale device. Moreover, it does not provide flexibility for alternative culture protocols to be used during the production cycle required for the large-scale production of, for example, clean meat. [Background technology]

[0002] Another such example of a prior art device and system is described in WO 2020 / 222239 ('239 Application) to Aleph Farms, Ltd. The '239 Application discloses a culture system for structured meat products, but the system is limited to utilizing culture bags in which cells are grown on a scaffold suspended within a bioreactor. Additionally, the '239 Application system is directed to a complex system requiring individual peristaltic pumps for each reactor and culture bioreactor that further require being rotated on their axes to reverse fluid flow. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent No. 8,492,140 [Patent Document 2] International Publication No. 2020 / 222239 Summary of the Invention [Problem to be solved by the invention]

[0004] What is needed in the art is a processing system designed for the production of clean meat products that is easy to set up, scalable and flexible, and enables cost-effective production of clean meat. [Means for solving the problem]

[0005] The present invention solves this need by providing a closed, continuous, semi-continuous or batch culture system for cell growth and differentiation followed by tissue growth for, for example, clean meat production. The process and system of the present invention solves this problem by utilizing only one bioreactor for cell growth and proliferation, first growing the cells and then expanding them in a perfusion type cycle and medium exchange. Once the cells are grown and expanded, the bioreactor is used as a medium supply vessel. The cells are removed from the bioreactor and, optionally separated from the medium by a cell medium separation device, are then grown to confluence to form tissue in a cell differentiation and tissue formation device, preferably with a scaffold suitable for cell attachment.

[0006] Thus, the present invention provides one or more of the following advantages over the prior art: reduced capital investment, disposable, long term use, ease of recovery, provides closed processing (with concomitant reduced chance of contamination), does not require physical cell transfer outside of a closed system, is easily scalable (up to 10,000 liters and beyond), utilizes a single bioreactor for multiple functions. The bioreactor may be a stirred cell bioreactor.

[0007] In one aspect, the invention provides a closed environment process for culturing cells to confluency and forming tissues, the process comprising the steps of providing a system comprising a cell growth and proliferation reactor, one or more tissue formation reactors, and optionally a cell retention device; seeding the cell growth and proliferation reactor and growing cells in the cell growth and proliferation reactor to a desired cell density; once the desired cell density is obtained, optionally processing the cells through the cell retention device, thereby transferring the cells to one or more tissue formation reactors and removing the growth medium; converting the bioreactor into a medium reservoir (e.g., a differentiation medium reservoir or a cell growth medium reservoir) for feeding the tissue growth reactors; differentiating and growing the cells in the one or more tissue formation reactors until a desired level of confluency is reached and tissue is formed, and harvesting tissue from the one or more tissue formation reactors.

[0008] In another aspect of the invention, the treatment system is semi-continuous or continuous.

[0009] In another embodiment of the invention, the cell growth and proliferation reactors range in size from 0.5 liters to 10,000 liters and 20,000 liters.

[0010] In another aspect of the invention, the cell growth and proliferation reactor has a size of 0.5 liters to 2000 liters.

[0011] In another aspect of the invention, the process further comprises a manifold system for integrating said tissue formation reactors when said processing system comprises more than one said tissue formation reactor.

[0012] In another aspect of the invention, the process further comprises one or more monitoring systems for i) dissolved oxygen, ii) pH, iii) carbon dioxide, iv) cellular waste products, v) one or more cellular metabolic products, vi) temperature, vii) flow rate, viii) cell density, and ix) cell viability.

[0013] In another aspect of the invention, the process further comprises bypassing the cell retention device.

[0014] In another aspect of the invention, the process further comprises one or more tissue formation reactors being hollow fiber reactors.

[0015] In another aspect of the invention, tissue can be withdrawn (aseptically or cleanly) from one or more of the one or more tissue formation reactors while maintaining the sterility of the remainder of the system.

[0016] In another embodiment, the retrieved tissue formation reactor can be sterilized and reseeded without compromising the integrity of the rest of the system.

[0017] In another aspect of the invention, the cells in the bioreactor are adapted for suspension growth, aggregate growth, or microcarrier growth.

[0018] In another aspect of the invention, one or more of the tissue formation reactors comprises a scaffold for cell attachment.

[0019] In another aspect, the invention comprises a closed environment process for culturing cells to confluency and forming tissue, the process comprising the steps of: a) providing i) a cell growth and proliferation reactor; ii) one or more tissue formation reactors; and iii) a cell retention device; b) i) seeding the cell growth and proliferation reactor and expanding the cell density in the cell growth and proliferation reactor; ii) once a desired cell density is obtained, iii) processing the cells through the cell retention device, thereby transferring a portion of the cells to one or more tissue formation reactors and transferring a portion of the cells back to a bioreactor; and iv) continuing to transfer the cells from the cell growth and proliferation reactor once a suitable cell density is available in the cell growth and proliferation reactor; and c) i) differentiating and growing the cells in the one or more tissue formation reactors until a desired level of confluency is reached and tissue is formed; and ii) harvesting tissue from the one or more tissue formation reactors.

[0020] In another aspect of the invention, the process further comprises a first reservoir holding a cell growth medium and a second reservoir holding a differentiation medium, wherein the cell growth medium is delivered to a cell growth and proliferation reactor and the differentiation medium is delivered to one or more tissue formation reactors after transferring the cells to the one or more tissue formation reactors.

[0021] In another aspect of the invention, the treatment system is semi-continuous or continuous.

[0022] In another aspect of the invention, the cell growth and proliferation reactor has a size of 0.5 liters to 20,000 liters.

[0023] In another aspect of the invention, the cell growth and proliferation reactor has a size of 0.5 liters to 2000 liters.

[0024] In another aspect of the invention, the process further comprises a manifold system for integrating said tissue formation reactors when said processing system comprises more than one said tissue formation reactor.

[0025] In another aspect of the invention, the process further comprises one or more monitoring systems for i) dissolved oxygen, ii) pH, iii) carbon dioxide, iv) cellular waste products, v) one or more cellular metabolic products, vi) temperature, vii) flow rate, viii) cell density, and ix) cell viability.

[0026] In another aspect of the invention, the process further comprises allowing the cell retention device to be bypassed.

[0027] In another embodiment of the invention, one or more of the tissue formation reactors is a hollow fiber reactor.

[0028] In another aspect of the invention, tissue can be aseptically withdrawn from one or more of the one or more tissue formation reactors while maintaining the sterility of the remainder of the system.

[0029] In another embodiment, the retrieved tissue formation reactor can be sterilized and reseeded without compromising the integrity of the rest of the system.

[0030] In another aspect of the invention, the cells in the bioreactor are adapted for suspension growth, aggregate growth, or microcarrier growth.

[0031] In another aspect of the invention, one or more of the tissue formation reactors comprises a scaffold for cell attachment.

[0032] Another aspect of the invention further comprises a separate reservoir for differentiation medium fluidly connected to said tissue formation reactor. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 illustrates one embodiment of the present invention. [Diagram 2] FIG. 2 shows a closed continuous or semi-continuous culture system of the present invention during the cell expansion (cell growth and proliferation) step of the process of the present invention. [Diagram 3] FIG. 3 shows a closed continuous or semi-continuous culture system of the present invention during the differentiation stage of the process of the present invention. [Figure 4] FIG. 4 is a diagram of a closed continuous or semi-continuous culture system of the invention during the loading step of the process of the invention, where the tissue forming bioreactor is seeded with cells from a bioreactor. [Diagram 5] FIG. 5 shows a closed continuous or semi-continuous culture system of the present invention during the growth or tissue generation stage of the process to produce the desired tissue. [Figure 6] FIG. 6 shows a schematic diagram of a prior art processing system that utilizes a stirred batch reactor used as the production vessel and a continuous seeded row reactor to increase the cell population prior to seeding. [Figure 7]FIG. 7 shows a schematic diagram of a processing system of the present invention in which a cell growth reactor (bioreactor: 2) is used as a medium reservoir after cells are seeded into a tissue formation reactor 6. [Figure 8] FIG. 8 shows a schematic diagram of a processing system of the present invention in which a cell growth reactor (bioreactor: 2) is used to produce multiple batches of cells (i.e., two or more batches of cells) for seeding multiple (i.e., two or more) tissue formation reactors in turn. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] FIG. 1 shows one embodiment of the present invention. 1 is growth medium containing cells. 2 is a bioreactor (i.e., growth and proliferation reactor). 3 is an optional culture parameter sampling device. 4 is an optional cell retention device. 5 is a manifold for selectively redirecting medium and cells between tissue formation reactors. 6 is three tissue formation reactors. 7 is a medium input line for directing medium to the ends of the tissue formation reactors. 8 is a cell seeding line. 9 is an outlet line from the tissue reactor central tube. 10 is an outlet line from the cell culture chamber of the tissue formation reactor. 12 is the impeller of the bioreactor / medium tank. One or more waste lines for removing spent medium are not shown. The waste lines can be located anywhere between the outlet lines and the bioreactors.

[0035] Figure 2 shows a closed continuous or semi-continuous culture system of the invention during the cell expansion (cell growth and proliferation) step of the process of the invention. Cells or medium are not directed to the tissue formation reactor. Cells are growing and proliferating in the bioreactor. Figures 2-5 also show different embodiments of the impeller 12 in the tank 2.

[0036] 3 shows a closed continuous or semi-continuous culture system of the invention during the differentiation stage of the process of the invention. The medium type is changed from growth medium to differentiation medium. In other embodiments, cells may be partially or fully differentiated in the tissue formation reactor.

[0037] FIG. 4 is a diagram of a closed continuous or semi-continuous culture system of the invention during the input step of the process of the invention, where a tissue formation bioreactor is seeded with cells from a bioreactor.

[0038] 5 shows a closed continuous or semi-continuous culture system of the present invention during the growth or tissue generation stage of the process to produce a desired tissue. In some embodiments, the cells are allowed to differentiate or continue to differentiate in the tissue formation reactor. In other embodiments, the cells are fully differentiated in the bioreactor when they are input into the tissue formation reactor.

[0039] FIG. 6 shows a schematic diagram of a prior art processing system utilizing a stirred batch reactor used as the production vessel and a continuous seeded row reactor to increase the cell population prior to seeding.

[0040] 7 shows a schematic diagram of a processing system of the present invention in which a cell growth reactor (bioreactor: 2) is used as a media reservoir after cells are seeded into a tissue formation reactor 6. In this embodiment of the present invention, the tissue formation reactor also functions as a differentiation reactor in which differentiation factors 15 are added to the cells within the tissue formation reactor.

[0041] FIG. 8 shows a schematic diagram of a processing system of the present invention in which a cell growth reactor (bioreactor:2) is used to produce multiple batches of cells (i.e., two or more batches of cells) for seeding multiple (i.e., two or more) tissue formation reactors in turn. After one tissue formation reactor row is harvested (three tissue formation reactor rows are shown) while maintaining the sterile integrity of the rest of the system, the harvested reactor can be sterilized and reseeded with cells from the bioreactor. In this system, a separate media reservoir is used to feed the tissue formation reactors. 14 is a media storage tank for feeding the tissue formation reactors.

[0042] The present invention relates to a closed environment process for culturing cells to confluence and forming tissues. In one embodiment, it is contemplated that the process comprises one or more cell growth and proliferation reactors, one or more tissue formation reactors, and optionally one or more cell retention devices.

[0043] In the present invention, a "cell growth and proliferation reactor" is defined as a bioreactor suitable for seeding one or more cell types and for maintaining and adjusting culture conditions to achieve a desired rate for cell growth and proliferation to reach a desired density or confluency. "Maintaining and adjusting" culture conditions is defined herein to mean adjusting the physical parameters required for the desired cell growth to a set value or range of values ​​and, if necessary, adjusting the parameters to achieve or maintain the desired cell growth rate. Such parameters may be, for example, but are not limited to, one or more of temperature, dissolved gas levels (e.g., oxygen and / or carbon dioxide), pH, cell waste products (e.g., lactic acid), one or more cell metabolic products, flow rate, cell density, and cell viability. It is contemplated that the cell growth and proliferation reactor is adapted for suspension growth, aggregate growth, or microcarrier growth. Cells may be partially or fully differentiated in the cell growth and proliferation reactor.

[0044] Furthermore, in the present invention, a "tissue formation reactor" is defined as a bioreactor specifically designed to enable and enhance the formation of desired tissues, and in some embodiments, the differentiation of cells, preferably cells grown and propagated within the "cell growth and proliferation reactor" of the present invention, to reach a density reminiscent of natural tissue. Such a reactor may be composed of an external tube with upper and lower end caps. The end caps and tubes have different inlets and outlets to allow for forward and reverse circulation of cells and medium. A smaller tube, with a defined porosity, is fixed between the upper and lower end caps inside the external tube. Fluid circulation is possible through this central tube, in both ways. The materials used in the assembly of the device may be specific grades of plastic (food grade, pharmaceutical grade), metal (e.g., stainless steel), or alternative materials known to those skilled in the art and compliant with food industry standards.

[0045] The tissue formation reactor may further comprise a scaffold suitable for cell attachment and / or growth. Such scaffolds are known to those skilled in the art and include hollow fibers, three-dimensional lattices, woven or non-woven materials, and the like.

[0046] Furthermore, in the present invention, a "cell retention device" is a device or system, such as a filtration system, that is specifically designed or adapted to allow separation of cells (e.g., cells grown and propagated in the "cell growth and proliferation device" of the present invention) from the liquid in which the cells are grown and propagated (e.g., culture medium) or other liquid in which the cells are placed (e.g., cell-compatible saline or buffer). The cell retention device filters the cells from the culture medium or other liquid. One purpose in this regard is to eliminate the "spent" medium (i.e., remove the cells from the "spent" medium). The cells are then resuspended in fresh medium. Another purpose is to change one type of medium to another. This may be necessary as the cells grow and proliferate, resulting in a denser culture that requires different medium components and / or different concentrations of medium components. Yet another purpose is to concentrate the cells to a higher concentration, for example, for effective seeding into the "tissue formation reactor" of the present invention. Yet another purpose of the cell retention device is to separate the cells from cell clusters or aggregates. The cell retention device of the present invention may perform any or all of these functions alone or simultaneously. The cell retention device of the present invention may perform these functions continuously or intermittently, and / or for some or all of the cells from the cell growth and proliferation device. The cell retention device may be used during certain steps (but not all steps) in the growth and differentiation of cells and generation of tissues. For example, the cell retention device may be used to remove cell aggregates prior to seeding into a tissue formation reactor, but not when the cells are returned to the cell growth and proliferation reactor (e.g., during medium exchange in the cell growth and proliferation reactor). The "cell retention device" of the present invention may be a stand-alone device in fluid communication with the cell growth and proliferation device, or may be integral with the "cell culture and proliferation device" and / or the "tissue formation reactor". In one embodiment, the cell retention device includes one or more tangential flow filters (TFF) or single pass tangential flow filters (SPTFF) or other filtration or screening mechanisms.

[0047] The present invention also contemplates a process for growing, proliferating and differentiating cells to form tissues using one or more of the cell growth and proliferation reactors, one or more of the tissue formation reactors and, optionally, the cell retention device of the present invention, which in one embodiment comprises the steps of seeding the cell growth and proliferation reactor and proliferating cells in the cell growth and proliferation reactor to a desired cell density, optionally processing the cells through a cell retention device once the desired cell density is achieved, then transferring the cells to one or more tissue formation reactors, removing growth medium from the bioreactor and converting the bioreactor into a differentiation medium reservoir for feeding the tissue growth reactors, differentiating and growing the cells as required in the one or more tissue formation reactors until a desired level of confluency is reached and tissue is formed, and recovering tissue from the one or more tissue formation reactors.

[0048] "Seeding" a bioreactor, as used herein, refers to the introduction of a low density of cells (e.g., 1×10 4 / ml~1×10 8 Cell "expansion" is defined herein as inoculating a total number of cells per unit volume (typically cells per milliliter (ml)) until a desired cell density is achieved. Once inside the bioreactor, the cells reproduce and grow / increase the population. Thus, cell "expansion" is defined herein as increasing the total number of cells per unit volume (typically cells per milliliter (ml)) until a desired cell density is achieved.

[0049] "Desired cell density" will vary depending on the cell type being cultured (some cell types do not grow to as high a density as others) and the end use of the cells. One of skill in the art, armed with the teachings herein, will be able to determine the desired cell density for a particular purpose.

[0050] In some embodiments, cells may be grown to confluency. For attachment-dependent cells (including cells grown on microcarriers), "confluency" is defined herein as covering at least 80%, 85%, 90%, 95%, 98%, 99% or 100% of the available surface area. For suspension cells, confluency is less well defined in the art, but is generally defined herein as approximately 1×10 9 ~1×10 12 Defined as cells / ml.

[0051] "Cell / cytoplasmic differentiation" is defined herein as the process by which cells change from one cell type to another. Usually, cells change into more specialized types. For example, during the development of an organism, stem cells differentiate into the specialized cell types that make up the organism. Induced pluripotent stem cells (iPSCs) are a type of stem cell that can be generated directly from somatic cells. iPSC technology was developed in 2006 by Shinya Yamanaka's laboratory in Kyoto, Japan, where Yamanaka showed that the introduction of four specific genes (Myc, Oct3 / 4, Sox2 and Klf4) that code for transcription factors can convert somatic cells into pluripotent stem cells (Takahashi K., Yamanaka S., August 2006, "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors," Cell, 126(4):663-676).

[0052] Stem cells and iPSCs can be differentiated into specialized cells (muscle, neural, fat, epithelium, etc.) by exposing the cells to specific differentiation factors. Stem cells and induced pluripotent stem cells can be induced to differentiate into a specific desired cell type or into cells with characteristics of a specific desired cell type. A specific cell type characteristic means that the cells exhibit, for example, morphological and molecular markers (e.g., cell surface or cytoplasmic markers) that are characteristic of or indicative of a specific cell type. For example, cells with muscle cell characteristics may exhibit one or more molecular markers such as PAX7, MYF5, MYOD1, and MYOG (see, for example, M. Shelton et al., Methods 101 (2016) 73-84). Cells with adipocyte characteristics may exhibit one or more of the following molecular markers: BMP4, Hox8, Hoxc9, Hoxc5 in white adipocyte progenitors and PRDM16, Dio2 and Pax3 in brown adipocyte progenitors (see, e.g., Mohsen-Kanson et al., Stem Cells. 2014 Jun;32(6):1459-67). It is known in the art what morphological and physiological markers and characteristics can be used to identify or are associated with a particular cell type. Muscle cells have been generated from iPSCs by those skilled in the art. See, e.g., M. Shelton et al., Methods 101 (2016) 73-84; Laine et al., Skeletal Muscle (2018) 8:1, both of which are incorporated herein in their entirety. Adipocytes have been generated from iPSCs, for example, by exposure to Oct4, Sox2, Klf4 (see, for example, Mohsen-Kanson et al., Stem Cells. 2014 Jun;32(6):1459-67, incorporated herein in its entirety). The morphological characteristics of muscle cells, adipocytes and other cells / tissues are well known to those of skill in the art. "Exposure" to a factor, as used herein, refers to the addition of the factor to the medium and / or the transfection of cells with a construct expressing the desired factor and / or the transfection of cells with a construct expressing a transcription factor that allows for the activation and inactivation of differentiation factors or factors that cause the cells to differentiate.

[0053] In the present invention, cells are differentiated to form one or more desired cell types. The cells may be at least partially differentiated in a tissue formation reactor of the present invention. In this regard, the cells may be first induced to differentiate in a cell growth and proliferation reactor of the present invention, if desired. Once a desired percentage of cells have differentiated (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or any percentage of cells from those listed herein), the cells are grown to confluence to form tissue. Confluence as used herein is defined above. In another embodiment, the cells are differentiated in a cell growth and proliferation reactor and then transferred to a tissue formation reactor. This procedure may be optimal for non-anchorage-dependent cells. In yet another embodiment, a portion of the cells are differentiated in a cell growth and proliferation reactor and a portion of the cells are differentiated in a cell differentiation and tissue formation reactor. In this embodiment, the percentage of differentiated cells in the cell growth and proliferation reactor may be 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or any percentage of cells from the percentages recited herein.

[0054] A "tissue" is defined herein as an ensemble of primarily similar cells (and sometimes their extracellular matrix) from the same or similar origin that together perform a particular function. Tissues are typically made up of primarily similar cells (e.g., muscle is made up primarily of muscle cells that form into myofibrils), but may include other cell types. For example, muscle tissue, in addition to muscle cells, often also comprises adipocytes, fibroblasts, nerve cells, etc.

[0055] The processing system of the present invention may be used to efficiently and economically produce structured clean meat products. Prior art systems (see, e.g., FIG. 6) are unable to efficiently or economically produce structured meat products that adequately meet any of the criteria presented below.

[0056] "Clean meat" is defined in the art as meat or meat-like products (collectively referred to herein as "clean meat" or "clean meat products") that are grown from cells in a laboratory, factory or other production facility suitable for the large-scale cultivation of cells.

[0057] A "structured meat product" or "structured clean meat product" is a meat product or clean meat product that has a texture and structure similar to or suggestive of natural meat of animal origin. The structured meat products of the present invention have a texture and structure similar to natural meat: 1) in texture and appearance, 2) in handleability when prepared for cooking and consumption (e.g., sliced, ground, cooked, etc.), and 3) in mouthfeel when consumed by humans.

[0058] A "closed environment" as defined herein refers to a system or culture system that does not expose the cells, culture medium or culture atmosphere to the outside atmosphere. In comparison, open culture systems are exemplified by petri dishes, culture flasks or microtiter plates. They expose the internal contents to the outside atmosphere as gas exchange occurs by diffusion from under the lid or cap of the culture vessel. The sterility of these culture systems depends on controlling the airflow around the vessel so that particles and other contaminants are not forced through the labyrinth through which gas must flow for proper gas exchange. Culture medium is usually exchanged manually on a benchtop (sometimes in a stationary hood to block airflow) or in a sterile filtered laminar flow hood. In contrast, in a closed environment, all gas exchange occurs through filtered ports and culture medium exchange occurs between sterile and fluidly connected supply and waste containers.

[0059] The present invention also contemplates that the processing system is a semi-continuous or continuous process. The term "continuous process" as used herein refers to a process for growing and differentiating cells that includes two or more process steps (or unit operations) such that the discharge from one process step flows directly to the next process step in the process without interruption and / or without the need to collect the entire volume of the discharge from the process step before the next process step is performed. In a preferred embodiment, two or more process steps can be performed simultaneously for at least a portion of their duration. In other words, in the case of continuous processes, as described herein, there is no need to complete a process step before the next process step is started, and a portion of the sample is always moving through the process steps. The term "continuous process" also applies to steps within a process operation, where during the execution of a process operation that includes multiple steps, the sample flows continuously through the multiple steps necessary to perform the process operation. One example of such a process operation, as described herein, is a flow-through cell culture operation that includes multiple steps that are performed continuously and that uses at least one cell growth and proliferation reactor, one or more of cell differentiation and tissue formation reactors, and optionally one or more cell retention devices.

[0060] The continuous processes described herein also include processes in which the input or output of fluid materials in any single process step is discontinuous or intermittent. Such processes may also be referred to as "semi-continuous" or "fed-batch" processes. For example, in certain embodiments according to the present invention, the inputs (e.g., cell seeding or medium transfer) in a process step may be loaded in a continuous or semi-continuous manner. Furthermore, the outputs, i.e., harvests, may be performed intermittently. Thus, in some embodiments, the processes and systems described herein include at least one unit operation that is operated in a semi-continuous or intermittent manner, while other unit operations in the process or system may be operated continuously.

[0061] The term "connected process" refers to a process for growing and differentiating cells, which comprises two or more process steps (or unit operations) connected in direct fluid communication with each other, such that fluid materials flow continuously or semi-continuously through the process steps within the process and are in contact with the two or more process steps simultaneously during normal operation of the process. It is understood that sometimes at least one process step within a process may be temporarily isolated from the other process steps by a barrier, such as a valve in a closed position. This temporary isolation of an individual process step may be necessary, for example, during start-up or stop of a process or during removal / replacement of an individual unit operation. The term "connected process" also applies to steps within a process operation that are connected in fluid communication with each other, for example, when the process operation needs to perform several steps to achieve the intended result of the operation (e.g., cell growth, proliferation and differentiation processes used in the methods described herein).

[0062] The present invention is not limited by the size of the cell growth and proliferation reactor. When used in accordance with the teachings herein, any available size reactor may be used in the present invention. In one embodiment, the cell growth and proliferation reactor is 0.1-20,000 liters, 0.1-10,000 liters, 0.5-5,000 liters, 0.5-2,000 liters, 0.5-1,000 liters, 0.5-800 liters, 0.5-500 liters, 0.5-300 liters, 0.5-100 liters, and 0.5-20 liters. Additionally, the cell growth and proliferation reactor may be any size within any of the above ranges.

[0063] Additionally, the present invention is not limited by either the number or size of tissue formation reactors. The size of the tissue formation reactor may depend, for example, on the desired size of the tissue to be produced, the physical limitations necessitated by the growth of cells to confluence, availability of reactors, etc. Similarly, the present invention is not limited to any particular number of tissue formation reactors. In one embodiment, the present invention contemplates 1, 2, 3, 4, 5, 10, 25, 50, 75, 100 or more reactors in one processing system, or any number of reactors between the specifically recited numbers, as desired by one of skill in the art. Multiple tissue formation reactors may be seeded simultaneously, in parallel, or sequentially with cells from a cell growth and proliferation reactor (i.e., cell differentiation from one and feeding the next by overflow from the tissue formation reactor). Similarly, tissue formation reactors may be harvested simultaneously or sequentially. When operated in series (seeded and harvested at confluence), the cell growth and proliferation reactor continuously supplies cells to newly installed tissue formation reactors, either as new locations when they are incorporated into the system, or as replacement reactors for harvested reactors. In this scenario, the cell growth and proliferation reactor is not converted into a container for differentiation medium. The cell differentiation and tissue formation reactors may receive medium from the cell growth and proliferation reactor, for example, after passing the cells and medium through a cell retention device, returning a portion of the cells and a portion of the medium to the cell growth and proliferation reactor, and returning a portion of the cells and medium to the tissue formation reactor. In this case, the cells in the cell growth and proliferation reactor and the tissue formation reactor utilize the same medium. In another scenario, additional components may be added to the medium after it is separated in the cell retention device, before it is fed to the tissue formation reactor, to supplement the medium coming from the cell growth and proliferation reactor. In yet another scenario, additional components may be added directly to the tissue formation reactor, to supplement the medium coming from the cell growth and proliferation reactor. In yet another scenario, separate containers may be used to supply differentiation medium and / or growth medium to the tissue formation reactor. Differentiation medium is a cell culture medium used to induce stem cells (e.g., iPSCs) to differentiate into a desired cell type or into cells having characteristics of a desired cell type.

[0064] When two or more tissue formation reactors are used, the system may optionally utilize a manifold system for directing media and other components to the reactors. Additionally, a manifold system may be used to isolate any one or more reactors for recovery and replacement (or other operations) and to maintain the integrity (e.g., sterility) of the remaining system components. The manifold system may be operated manually or may be automated or semi-automated. Control systems, including computer control systems that automate the manifold or other portions of the processing system, are also embodied by the present invention and are described in more detail below.

[0065] The processing system of the present invention may also include a monitoring system for monitoring and analyzing the culture conditions and medium. The monitoring system may include one or more systems (including sensors and probes) for measuring i) dissolved oxygen, ii) pH, iii) carbon dioxide, iv) cellular waste products, v) one or more cellular metabolic products, vi) temperature, vii) flow rate, viii) cell density, and ix) cell viability. Suitable sensors and probes are known to those of skill in the art. Reactor conditions may be monitored within the cell growth and proliferation reactor, within a sampling chamber fluidly connected to the cell growth and proliferation reactor, within one or more of the tissue growth reactors, within a sampling chamber fluidly connected to one or more of the tissue formation reactors, or within any other part of the system where one of skill in the art would understand that a sample representative of the culture conditions within the system may be obtained.

[0066] In an embodiment, sensors and / or probes may be connected to a sensor electronics module, the output of which may be transmitted to a terminal board and / or junction box. The results of the sensing operation may be input to a computer-implemented control system (e.g., a computer) for calculation and control of various parameters (e.g., temperature, pH, dissolved gases) and for display and user interface. Such a control system may also include a combination of electronic, mechanical, and / or pneumatic systems for controlling process parameters. It should be understood that the control system may perform other functions and the invention is not limited to having any particular function or set of functions.

[0067] In certain embodiments of the invention, a cell retention device may be utilized to separate cells from the medium. This may be desirable, for example, when cells are transferred from a cell growth and proliferation reactor to a tissue formation reactor. A cell retention device need not be required in each and every embodiment of the invention or used during every step in a process cycle. For example, in some embodiments, a cell retention device may be present but bypassed. In other embodiments, the cell retention device may be eliminated entirely. In processes of the invention where the cell retention device is bypassed or eliminated, the function of the cell retention device, i.e., separation of cells and medium, may be performed, for example, by either the cell growth and proliferation reactor and / or the tissue growth reactor. For example, when cells from a cell growth and proliferation reactor are seeded into a tissue growth reactor, the cells are retained by the tissue growth reactor and the medium can be directed, for example, to a waste vesicle.

[0068] The tissue formation reactor of the present invention may be any device suitable for differentiation and / or growth of cells into a desired tissue. Suitable reactors known in the art include, but are not limited to, hollow fiber reactors and other types of scaffold-equipped reactors known to those skilled in the art suitable for cell attachment and growth.

[0069] The treatment system of the present invention is not directed to the culture of any particular cell type. Preferably, undifferentiated or dedifferentiated cells are utilized and differentiated in the system. However, the treatment system of the present invention may also be utilized for the culture of differentiated cells.

[0070] Cell culture parameters are determined by the cell type being cultured. Cell culture parameters include, but are not limited to, medium, additional medium components, medium exchange rate, temperature, pH, gas exchange rate, etc. Furthermore, cell culture parameters may change as cells differentiate and grow. For example, during differentiation, specific growth factors may be required. During proliferation, greater amounts of medium exchange and / or gas exchange may be required. With the guidance of this specification, one skilled in the art will be able to determine the cell culture parameters for the cell type being cultured.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0072] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0073] As used herein, the transitional phrases "comprising," "consisting essentially of," and "consisting of" have the meaning set forth in MPEP 2111.03. Any claim using the transitional phrase "consisting essentially of" shall be understood to recite only essential elements of the invention. Any claim dependent on a claim reciting "consisting essentially of" shall be understood to recite elements that are not essential to the invention.

[0074] All ranges include all values ​​within the cited range, including all integers, fractions and decimals.

[0075] This invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications, and figures cited throughout this application are hereby incorporated by reference.

[0076] Example Example 1 The processing system of the present invention may be run in batch, fed-batch and continuous modes. This example illustrates running the processing system of the present invention in batch mode. The processing system of the present invention may be used, for example, to produce structured meat products, the process of which is illustrated herein.

[0077] The processing system is configured and connected essentially as shown in Figure 1. The processing system comprises at least a cell growth and proliferation reactor 2, a tissue growth reactor 6, and optionally a cell retention device 4. In view of the teachings herein, other configurations can be envisioned and utilized by those of skill in the art and are included herein.

[0078] Proper installation of the growth and proliferation reactors, cell retention device and tissue formation reactor is completed, including making the necessary sterile connections. In one embodiment, there can be two or more growth and proliferation reactors. Disposable reactor bags are used in one or more of the growth and proliferation reactors. Sensors (e.g., item 3) are connected and culture control parameters are established and entered into a control device (e.g., computer).

[0079] Batch mode involves adding medium and inoculum (approximately 1 × 10 6 The process consists of filling the bioreactor with culture medium (at 2000 cells / ml of seed cell suspension), operating it at predetermined parameters, and adjusting the bioreactor and / or medium as needed or indicated through sensors including pH (approximately 6.8-7.3), carbon dioxide (approximately 5%), oxygen, temperature (approximately 37 °C), etc.

[0080] The medium used in this step is defined for cell growth and may therefore be serum-based, serum-free or xeno-free. Xeno-free medium is defined herein to mean a formulation that is composed only of components derived from a single organism (e.g., bovine, porcine, etc.) and does not incorporate components derived from foreign species. Xeno-free medium may or may not be serum-free. Components may be naturally derived or engineered. Those skilled in the art can select a medium suitable for the culture cell type following the guidance of this specification.

[0081] The cells used to seed the bioreactor in this example are iPSCs, but may be any desired cells. The cells may be suspension or adherent cells. In the case of adherent cells, it is desirable to use a screen or other device at the outlet of the bioreactor to limit the size of the cell aggregates. This helps to create a uniform culture in the bioreactor. The screen is used to measure the aggregates and limit the size of the aggregates to allow good flow of the medium and therefore nutrients to the cells (if the aggregates are too large, the cells inside the aggregates will not survive as they will not get any nutrients from the medium). The screen can be placed at the outlet of the bioreactor before the retention system or just after the retention device on the recirculation loop to the bioreactor.

[0082] Cells may be differentiated in either the growth and proliferation reactor 2 or the tissue formation reactor 6. This will depend, at least in part, on the cell type being cultured. For example, to avoid the step of detaching cells from surfaces within the cell growth and proliferation reactor, it is preferable to differentiate cells that are adherent after loading into the tissue formation reactor.

[0083] When differentiating cells in a cell growth and proliferation reactor, once the cell growth profile is reached, the next step is to exchange the medium with specialized medium for the differentiation process using a recirculation circuit through the cell retention device and system. As with the cell growth stage of the culture, the medium may be serum-based or serum- or xeno-free. The medium may be the same as that used for growing the cells, or it may be specialized medium to induce differentiation of the cells into the desired cell type. In this example, the desired cell type is one or more of bovine muscle cells, bovine muscle cell-like cells, or cells engineered to have characteristics of bovine muscle cells.

[0084] In an alternative procedure, the cells are transferred to a tissue formation reactor prior to differentiation. As mentioned above, this is the preferred method for cells that adhere depending on the differentiation.

[0085] After the growth step, and, if desired, the differentiation step, the cells and medium are seeded into a tissue formation (and differentiation) reactor, e.g., a hollow fiber device. The cell density in the cell growth and proliferation reactor is approximately 1 x 10 9 ~1×10 12 cells / ml. The cells are transferred through a cell retention device, which separates the cells from the spent medium and optionally filters out cell aggregates. In batch mode, transfer continues until the total transfer of biomass from the bioreactor is complete. The bioreactor is then used as a medium container and continues to supply the cells in the tissue formation reactor until harvest. The cells are supplied with a medium suitable for growth (and, if necessary, differentiation) until the cells in the tissue formation reactor have grown to the desired cell type (e.g., myocytes or myocyte-like cells) and the final desired level of confluency and tissue structure (e.g., myofibrils that give an appearance and texture similar to natural meat) and are harvested. The spent medium is removed from the system after exiting the tissue formation reactor and can be partially or completely replaced by fresh medium.

[0086] Upon harvest, further processing of the structured cultured meat product occurs, as needed, including adding flavors, fats, and additional texture.

[0087] In this example, the final product is a cultured meat product that has an appearance, texture, handling and taste similar to natural meat, however, those of skill in the art, given the teachings herein, can use the processing system of the present invention to create other desired products.

[0088] Example 2 The processing system of the present invention may also be implemented in fed-batch and continuous modes. In fed-batch (semi-batch) mode, cells grown and expanded in a cell growth and proliferation reactor are intermittently delivered to one or more tissue formation reactors (Figures 1 and 8). In this processing system, the growth and proliferation reactor is not converted into a medium vessel. Rather, a separate vessel (see number 14 in Figure 8) is used as a medium vessel to feed the tissue formation reactor. Cell transfer is interrupted intermittently to allow for further cell growth and proliferation or reseeding, if necessary. Also in this mode, tissue formation reactors are continuously harvested as each one reaches confluency and is replaced with a new reactor. Figures 2-5 show the various steps in this embodiment of the present invention. Cell growth (Figure 2) circulates medium through a processing probe (number 3 in Figure 4) to monitor culture conditions and cell growth. The medium is replaced with differentiation medium (Figure 3) and the cells can be differentiated in the bioreactor. Once the correct cell density of differentiated cells is obtained, the cells are transferred to the tissue formation reactor, optionally after processing through a cell retention device. See FIG. 4. This may be referred to as the input step. FIG. 5 shows the tissue formation step, where the cells are grown to the desired confluency in the tissue formation reactor. FIG. 7 shows the differentiation factors being added to the tissue formation reactor from separate containers 15 for an embodiment in which differentiation occurs at least partially in the tissue formation reactor. FIG. 8 shows three banks of tissue formation reactors. These banks of reactors may be seeded at different times and therefore harvested and reseeded at different times, making the process a continuous process. The spent medium is removed from the system after exiting the tissue formation reactor and may be replaced completely or partially by fresh medium.

[0089] Continuous mode is similar to fed-batch mode, but cell growth and proliferation is at a rate that allows for continuous transfer of cells into the tissue formation reactor. In this mode, two or more cell growth and proliferation reactors can be used.

Claims

1. 1. A method of closed environment processing for culturing cells to confluence to form clean meat, comprising: a) providing a processing system comprising: i) a cell growth and proliferation reactor; ii) one or more tissue formation reactors; and iii) a cell retention device; b) i) seeding said cell growth and proliferation reactor and expanding the cell density in the cell growth and proliferation reactor to a desired cell density; ii) once the desired cell density is obtained, processing the cells through a cell retention device, thereby transferring the cells to one or more tissue formation reactors and removing the growth medium; and iii) using the cell growth and proliferation reactor as a differentiation medium reservoir to supply medium to the tissue formation reactors. c) i) differentiating and growing the cells in the one or more tissue formation reactors until a desired level of confluency is reached and clean meat is formed; and ii) harvesting the clean meat from the one or more tissue formation reactors. A method for treating an enclosed environment comprising:

2. The method of claim 1 , wherein the processing system is semi-continuous or continuous.

3. 3. The method of claim 1 or 2, wherein the cell growth and proliferation reactor is between 0.5 liters and 20,000 liters in size.

4. 4. The method of claim 3, wherein the size of the cell growth and proliferation reactor is between 0.5 liters and 2000 liters.

5. The method of any one of claims 1 to 4, further comprising, when the processing system comprises more than one tissue formation reactor, a manifold system for integrating the tissue formation reactors.

6. 6. The method of any one of claims 1 to 5, further comprising one or more monitoring systems for: i) dissolved oxygen, ii) pH, iii) carbon dioxide, iv) cellular waste products, v) one or more cellular metabolic products, vi) temperature, vii) flow rate, viii) cell density, and ix) cell viability.

7. The method of any one of claims 1 to 6, wherein the cell retention device can be bypassed.

8. The method according to any one of claims 1 to 7, wherein the one or more tissue formation reactors are hollow fiber reactors.

9. 9. The method of any one of claims 1-8, wherein clean meat can be aseptically withdrawn from one or more of the one or more tissue formation reactors while maintaining sterility of the remainder of the processing system.

10. The method of any one of claims 1 to 9, wherein the cells in the cell growth and proliferation reactor are adapted for suspension growth, aggregate growth or microcarrier growth.

11. The method according to any one of claims 1 to 9, wherein the one or more tissue formation reactors comprise a scaffold for cell attachment.

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