Processing system for bioreactor-based clean meat production

Abstract

To provide a processing system that enables the cost-effective generation of clean meat.SOLUTION: Provided is closed environment processing for the culture of cells to confluency to form tissue, the processing comprising: a) providing, a system comprising i) a cell growth and expansion reactor, ii) tissue formation reactors, and iii) a cell retention device; b) i) seeding the cell growth and expansion reactor and expanding the cell density within the cell growth and expansion reactor to a desired cell density, ii) processing the cells through the cell retention device thereby transferring the cells to the tissue formation reactors and removing the growth media, and, iii) converting the bioreactor to a differentiation media reservoir for feeding the tissue growing reactors; and c) i) differentiating and growing the cells in the tissue formation reactors until a desired level of confluency is reached and tissue formed, and ii) collecting the tissue from the tissue formation reactors.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] For example, efficient, closed continuous, semi-continuous, or batch cell and tissue culture systems are needed 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 benchtop, laboratory-scale device specifically designed for producing 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. Furthermore, 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 (the '239 Application) to Aleph Farms, Ltd. While the '239 Application discloses a culture system for structured meat products, 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 need to be 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 that enables the cost-effective production of clean meat. [Means for solving the problem]

[0005] The present invention addresses 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 address this issue by utilizing only one bioreactor for cell growth and expansion, 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, optionally separated from the medium by a cell-medium separation device, and then grown to confluence to form tissue in a cell differentiation and tissue formation device, preferably equipped 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, easy retrieval, 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 or more), utilizes a single bioreactor for multiple functions. The bioreactor may be a stirred cell bioreactor.

[0007] In one aspect, the present invention provides a closed environment process for culturing cells to confluency and forming tissue, 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 expanding the cells in the cell growth and proliferation reactor to a desired cell density; once the desired cell density is achieved, 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 processing system is semi-continuous or continuous.

[0009] In another aspect 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 present 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 has two or more said tissue formation reactors.

[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 collected (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 tissue formation reactors comprise 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: 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 achieved, iii) processing the cells through the cell retention device, thereby transferring a portion of the cells to one or more tissue formation reactors and a portion of the cells back to a bioreactor; and iv) continuing to transfer cells from the cell growth and proliferation reactor when an appropriate 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 processing system is semi-continuous or continuous.

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

[0023] In another aspect of the present 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 has two or more said tissue formation reactors.

[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 tissue formation reactors are hollow fiber reactors.

[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 tissue formation reactors comprise a scaffold for cell attachment.

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

[0033] [Figure 1] FIG. 1 shows one embodiment of the present invention. [Figure 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. [Figure 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, in which the tissue-forming bioreactor is seeded with cells from a bioreactor. [Figure 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 continuous seed train reactor to increase the cell population prior to inoculating a stirred batch reactor used as the production vessel. [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 sequentially seeding multiple (i.e., two or more) tissue formation reactors. DETAILED DESCRIPTION OF THE INVENTION

[0034] FIG. 1 illustrates 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 diverting 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 line and the bioreactors.

[0035] Figure 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. Cells or medium are not directed to the tissue formation reactor. Cells are growing and proliferating within the bioreactor. Figures 2-5 also show different embodiments of the impeller 12 within 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 a growth medium to a differentiation medium. In other embodiments, cells may be partially or fully differentiated within 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, in which a tissue-forming 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, cells may differentiate or continue to differentiate in the tissue formation reactor. In other embodiments, cells are fully differentiated within the bioreactor when introduced into the tissue formation reactor.

[0039] FIG. 6 shows a schematic diagram of a prior art processing system that utilizes a continuous seed train reactor to increase the cell population prior to inoculating a stirred batch reactor used as the production vessel.

[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] Figure 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 (a row of three tissue formation reactors is 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, the process is contemplated to comprise one or more cell growth and proliferation reactors, one or more tissue formation reactors, and optionally one or more cell retention devices.

[0043] For purposes of the present invention, a "cell growth and proliferation reactor" is defined as a bioreactor suitable for seeding one or more cell types and maintaining and adjusting culture conditions to achieve a desired rate of 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 the following: temperature, dissolved gas levels (e.g., oxygen and / or carbon dioxide), pH, cellular waste products (e.g., lactic acid), one or more cellular metabolic products, flow rate, cell density, and cell viability. It is contemplated that the cell growth and proliferation reactor may be adapted for suspension growth, aggregate growth, or microcarrier growth. Cells may be partially or fully differentiated within 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 expanded within the "cell growth and proliferation reactor" of the present invention, to reach densities reminiscent of native tissues. Such a reactor may consist of an outer tube with upper and lower end caps. The end caps and tubes have distinct inlets and outlets to allow for forward and reverse cell and media circulation. A smaller tube with defined porosity is fixed inside the outer tube between the upper and lower end caps. Fluid circulation is possible through this central tube in both directions. 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 nonwoven materials, and the like.

[0046] Furthermore, in the present invention, a "cell retention device" refers to a device or system, such as a filtration system, specifically designed or adapted to enable separation of cells (e.g., cells grown and propagated in a "cell growth and proliferation device" of the present invention) from a liquid in which the cells are grown and propagated (e.g., culture medium) or another liquid in which the cells are placed (e.g., a cell-compatible saline or buffer solution). 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 for another. This may be necessary as cells grow and proliferate, resulting in a higher density 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 a "tissue formation reactor" of the present invention. Yet another purpose of the cell retention device is to separate 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 a cell growth and expansion apparatus. The cell retention device may be used during certain steps (but not all steps) in cell growth and differentiation and tissue generation. For example, a 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 expansion reactor (e.g., during medium exchange in the cell growth and expansion reactor). The "cell retention device" of the present invention may be a standalone device fluidly connected to a cell growth and expansion apparatus, or may be integrated with a "cell culture and expansion apparatus" and / or a "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 cell growth and proliferation reactors, one or more tissue formation reactors, and optionally, a cell retention device of the present invention. The process of the present invention, in one embodiment, comprises seeding the cell growth and proliferation reactor and expanding the 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 supplying the tissue growth reactors, differentiating and growing the cells as needed 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 x 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 the desired cell density is achieved. Once in the bioreactor, the cells reproduce and the population grows / expands. Thus, cell "expansion" is defined herein as increasing the total number of cells per unit volume (typically cells per milliliter (ml)) until the desired cell density is achieved.

[0049] The "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 confluence. For anchorage-dependent cells (including cells grown on microcarriers), "confluence" is defined herein as covering at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the available surface area. For suspension cells, confluence 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 differentiation / cytoplasmic differentiation" is defined herein as the process by which cells change from one cell type to another. Typically, 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 the Shinya Yamanaka Laboratory in Kyoto, Japan, and Yamanaka demonstrated that the introduction of four specific genes encoding transcription factors (Myc, Oct3 / 4, Sox2, and Klf4) 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, adipose, epithelial, etc.) by exposing the cells to specific differentiation factors. Stem cells and induced pluripotent stem cells can be induced to differentiate into specific desired cell types or cells with characteristics of specific desired cell types. Characteristics of a specific cell type mean 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, e.g., M. Shelton et al., Methods 101 (2016) 73-84). Cells with adipocyte characteristics may exhibit one or more molecular markers, such as BMP4, Hox8, Hoxc9, Hoxc5 in white adipocyte precursor cells, and PRDM16, Dio2, and Pax3 in brown adipocyte precursor cells (see, for example, Mohsen-Kanson et al., Stem Cells. 2014 Jun;32(6):1459-67). Morphological and physiological markers and characteristics that can be used to identify or are associated with specific cell types are known in the art. Muscle cells have been generated from iPSCs by those skilled in the art. See, for example, 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 entireties). Adipocytes have been generated from iPSCs by exposure to, for example, Oct4, Sox2, or Klf4 (see, e.g., Mohsen-Kanson et al., Stem Cells. 2014 Jun;32(6):1459-67, incorporated herein in its entirety). The morphological characteristics of myocytes, adipocytes, and other cells / tissues are well known to those skilled in the art. "Exposure" to a factor, as used herein, refers to the addition of the factor to the culture 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 the activation and inactivation of differentiation factors or factors that cause cells to differentiate.

[0053] In the present invention, cells are differentiated to form one or more desired cell types. Cells may be at least partially differentiated in a tissue formation reactor of the present invention. In this regard, cells may first be 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 enumerated herein), the cells are grown to confluence to form tissue. Confluence, as used herein, is defined above. In another embodiment, 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 enumerated 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 specific function. Tissues are typically made up of primarily similar cells (e.g., muscle is made up primarily of myocytes that form into myofibrils), but may also include other cell types. For example, muscle tissue often comprises, in addition to myocytes, 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 fully 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 that resembles natural meat in 1) texture and appearance, 2) handleability when prepared for cooking and consumption (e.g., when sliced, ground, cooked, etc.), and 3) mouthfeel when consumed by humans.

[0058] As defined herein, a "closed environment" refers to a system or culture system in which cells, culture medium, or culture atmosphere are not exposed to the external atmosphere. By comparison, open culture systems are exemplified by Petri dishes, culture flasks, or microtiter plates. These expose their internal contents to the external atmosphere because gas exchange occurs by diffusion from under the lid or cap of the culture vessel. Sterility in these culture systems relies on controlled airflow around the vessel to prevent particles and other contaminants from being forced through the labyrinth through which gas must flow for proper gas exchange. Culture medium is typically exchanged manually on a benchtop (sometimes in a stationary hood to block airflow) or in a sterile, filtered laminar flow hood. In contrast, in closed environments, all gas exchange occurs through filtered ports, and medium exchange occurs between sterile, fluidly connected supply and waste containers.

[0059] The present invention also contemplates that the processing system may be a semi-continuous or continuous process. As used herein, the term "continuous process" refers to a process for growing and differentiating cells that includes two or more process steps (or unit operations) such that the output 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 output from the process step before performing the next process step. In preferred embodiments, two or more process steps can be performed simultaneously for at least a portion of their duration. In other words, with continuous processes, as described herein, a process step does not need to be completed before the next process step begins; 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 utilize at least one cell growth and proliferation reactor, one or more 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 are sometimes 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 media transfer) in a process step may be loaded continuously or semi-continuously. Additionally, 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 semi-continuously or intermittently, while other unit operations within the process or system may be operated continuously.

[0061] The term "connected process" refers to a process for growing and differentiating cells, comprising two or more processing steps (or unit operations) connected in direct fluid communication with each other, such that fluid materials flow continuously or semi-continuously through the processing steps within the process and simultaneously contact two or more processing steps during normal operation of the process. It is understood that, at times, at least one processing step within a process may be temporarily isolated from other processing steps by a barrier, such as a valve in a closed position. This temporary isolation of an individual processing step may be necessary, for example, during start-up or stoppage of the process or during removal / replacement of an individual unit operation. The term "connected process" also applies to steps within a processing operation that are fluidly connected to each other, for example, when the processing operation needs to perform several steps to achieve the intended result of the operation (e.g., the 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 / expansion reactor. When used in accordance with the teachings herein, reactors of any available size may be used with the present invention. In one embodiment, the cell growth / expansion reactor is 0.1 to 20,000 liters, 0.1 to 10,000 liters, 0.5 to 5,000 liters, 0.5 to 2,000 liters, 0.5 to 1,000 liters, 0.5 to 800 liters, 0.5 to 500 liters, 0.5 to 300 liters, 0.5 to 100 liters, and 0.5 to 20 liters. Additionally, the cell growth / expansion reactor may be any size within any of the above ranges.

[0063] Furthermore, 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 constraints imposed by growing cells to confluence, reactor availability, 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 a single processing system, or any number 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 / expansion reactor (i.e., cell differentiation from one reactor and feeding the next via overflow from the tissue formation reactor). Similarly, tissue formation reactors may be harvested simultaneously or sequentially. When operated continuously (seeded and harvested at confluence), the cell growth and proliferation reactor continuously supplies cells to newly installed tissue formation reactors as they are incorporated into the system, either as a new location or as a replacement reactor for the harvested reactor. In this scenario, the cell growth and proliferation reactor is not converted into a reservoir 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 to supplement the medium coming from the cell growth and proliferation reactor before it is fed to the tissue formation 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 cells with 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, the 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 manually operated or automated or semi-automated. Control systems, including computerized 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 culture conditions and media. 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 skilled 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 from which a skilled artisan would understand that a sample representative of the culture conditions within the system may be obtained.

[0066] In some embodiments, 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 present invention is not limited to having any particular function or set of functions.

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

[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, more extensive medium exchange and / or gas exchange may be required. Those skilled in the art will be able to determine the cell culture parameters for the cell type being cultured using the guidance of this specification.

[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 meanings 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 drawings cited throughout this application are hereby incorporated by reference.

[0076] Example Example 1 The processing system of the present invention may be operated in batch, fed-batch, and continuous modes. This example illustrates operating 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 exemplified 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 skilled in the art and are included herein.

[0078] Proper installation of the growth and proliferation reactor, 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., 3) are connected, and culture control parameters are established and entered into a control device (e.g., a computer).

[0079] Batch mode involves adding medium and inoculum (approximately 1 × 10 6 The process consists of filling the bioreactor with the required amount of culture medium (cells / ml seed cell suspension), operating it at predetermined parameters, and adjusting the bioreactor and / or medium as needed or as 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 process is defined for cell growth and may therefore be a serum-based medium, a serum-free medium, or a xeno-free medium. Xeno-free medium is defined herein to mean a preparation composed solely 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 occurring or engineered. Those skilled in the art can select a medium appropriate for the culture cell type following the guidance herein.

[0081] In this example, the cells used to seed the bioreactor are iPSCs, but they can be any desired cells. The cells can be suspension or adherent cells. For adherent cells, a screen or other device is preferably used at the exit of the bioreactor to limit the size of cell aggregates. This helps create a uniform culture within the bioreactor. The screen is used to measure and limit the size of the aggregates to allow for good flow of medium and therefore nutrients to the cells (if the aggregates are too large, the cells inside the aggregates will not survive because they will not receive any nutrients from the medium). The screen can be placed at the exit of the bioreactor before the retention system or immediately 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 detaching cells from the surface 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 for a 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 cell growth, or it may be specialized 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 bovine muscle cell characteristics.

[0084] In an alternative procedure, cells are transferred to a tissue-forming reactor prior to differentiation. As mentioned above, this is the preferred method for cells that adhere upon 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 The cell volume is 1000 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 biomass transfer from the bioreactor is complete. The bioreactor is then used as a medium reservoir, continuing 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 they 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 provide 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 with fresh medium.

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

[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, one 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 can 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 reservoir. Rather, a separate vessel (see reference number 14 in Figure 8) is used as the medium reservoir to feed the tissue formation reactor. Cell transfer is intermittently interrupted to allow for further cell growth and expansion or reseeding, as needed. Also in this mode, tissue formation reactors are continuously harvested as each reaches confluence and replaced with a new reactor. Figures 2-5 illustrate various steps in this embodiment of the present invention. Cell growth (Figure 2) involves circulating 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 cells can be differentiated within the bioreactor. Once the correct cell density of differentiated cells is obtained, the cells are optionally processed through a cell retention device and then transferred to a tissue formation reactor. See Figure 4. This is sometimes referred to as the input step. Figure 5 illustrates the tissue formation step, in which cells are grown to a desired confluency in a tissue formation reactor. Figure 7 illustrates differentiation factors being added to the tissue formation reactor from separate containers 15 for embodiments in which differentiation occurs at least partially within the tissue formation reactor. Figure 8 illustrates 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 continuous. Spent medium is removed from the system after exiting the tissue formation reactor and may be completely or partially replaced with 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. A closed environment treatment for culturing cells to confluence and forming tissue, comprising: a) providing a 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 achieved, 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) converting the bioreactor into a differentiation medium reservoir for feeding the tissue growth 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 tissue is formed; and ii) harvesting tissue from the one or more tissue formation reactors; A closed environment treatment comprising:

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

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

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

5. The process of claim 1 , further comprising a manifold system for integrating said tissue formation reactors when said processing system has more than one said tissue formation reactor.

6. 10. The process of claim 1, 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 process of claim 1 , further comprising: allowing the cell retention device to be bypassed.

8. 10. The process of claim 1, wherein the one or more tissue formation reactors are hollow fiber reactors.

9. 10. The process of claim 1, wherein tissue can be aseptically retrieved from one or more of the one or more tissue formation reactors while maintaining sterility of the remainder of the system.

10. 10. The process of claim 1, wherein the cells in the bioreactor are adapted for suspension growth, aggregate growth, or microcarrier growth.

11. The process of claim 1 , wherein the one or more tissue formation reactors comprise a scaffold for cell attachment.

12. A closed environment treatment for culturing cells to confluence and forming tissue, comprising: a) providing i) a cell growth and proliferation reactor, ii) one or more tissue formation reactors, and iii) a cell retention device; b) i) inoculating said cell growth and proliferation reactor and expanding the cell density in the cell growth and proliferation reactor; ii) once a desired cell density is achieved, iii) processing the cells through a cell retention device, thereby transferring a portion of the cells to one or more tissue formation reactors and a portion of the cells back to the bioreactor; and iv) continuing to transfer cells from the cell growth and proliferation reactor to one or more tissue formation reactors once a suitable cell density is available in the cell growth and proliferation reactor; 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; A closed environment treatment comprising:

13. 13. The process of claim 12, further comprising 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.

14. 13. The process of claim 12, wherein the processing system is semi-continuous or continuous.

15. 13. The process of claim 12, wherein the cell growth and proliferation reactor is between 0.5 liters and 20,000 liters in size.

16. 16. The process of claim 15, wherein the cell growth and proliferation reactor is between 0.5 liters and 2000 liters in size.

17. 13. The process of claim 12, further comprising a manifold system for integrating said tissue formation reactors when said processing system has more than one said tissue formation reactor.

18. 13. The process of claim 12, 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.

19. 13. The process of claim 12, further comprising: allowing the cell retention device to be bypassed.

20. 13. The process of claim 12, wherein the one or more tissue formation reactors are hollow fiber reactors.

21. 13. The process of claim 12, wherein tissue can be aseptically withdrawn from one or more of the one or more tissue formation reactors while maintaining sterility of the remainder of the system.

22. 13. The process of claim 12, wherein the cells in the bioreactor are configured for suspension growth, aggregate growth, or microcarrier growth.

23. The process of claim 12 , wherein the one or more tissue formation reactors comprise a scaffold for cell attachment.

24. 13. The process of claim 12, further comprising a separate reservoir for differentiation medium fluidly connected to the tissue formation reactor.

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