Method for washing and polishing grown cell masses

Abstract

The present disclosure relates to a method of washing cells from a grown cell mass to remove cell culture medium and enrich the cells with a finishing medium. The disclosed method includes growing the cell mass in cell culture medium, then harvesting and washing the grown cell mass with a series of wash buffers or gradient wash buffers that change over time. Although cell culture medium contains nutrients and components that are beneficial for cell growth, grown cells in cell culture medium often have off-flavors, off-flavors, low color, low salt / mineral composition, and other deficiencies. Thus, the disclosed method includes removing cell culture medium remnants from the grown cell mass using a single or series of wash media. The grown cell mass is further washed with a finishing or enrichment buffer to further improve the sensory profile and nutritional composition of the grown cell mass.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 18 / 146,541, entitled "Method for Washing and Finishing a Grown Cell Mass," filed December 27, 2022, and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 294,703, entitled "Method for Washing and Finishing a Grown Cell Mass," filed December 29, 2021. This application also claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 294,700, entitled "Method for Pressurizing Cells Grown in Hydrogel to Induce Hypertrophy," filed December 29, 2021. The foregoing applications are incorporated herein by reference in their entireties. [Background technology]

[0002] As the world's population continues to grow, cell-based or cultured meat products for consumption have emerged as an attractive alternative (or supplement) to traditional meat derived from animals. For example, cell-based, cultured, or cultivated meat represents a technology that can address specific dietary needs of humans. Because the cells for cell-based meat are grown in the laboratory, laboratory methods of preparing cell-based meat can modify the profile of essential amino acids and fats, as well as enrich the meat in vitamins, minerals, and bioactive compounds. In some cases, cell-based meat products can be prepared from a combination of cultured adherent and suspension cells derived from non-human animals that facilitate such modifications and enrichments.

[0003] In addition to addressing dietary needs, cell-based meat products help alleviate some of the drawbacks associated with conventional meat products for humans, livestock, and the environment. For example, conventional meat production involves controversial practices associated with animal husbandry and slaughter. Other drawbacks associated with conventional meat production include low conversion of caloric inputs into edible nutrients, microbial contamination of the product, the occurrence and transmission of veterinary and zoonotic diseases, relative natural resource requirements, and resulting industrial pollutants such as greenhouse gas emissions and nitrogenous waste streams.

[0004] Despite the advances in making cell-based meat products, existing methods for culturing and processing cell-based meat products face several drawbacks, such as challenges or obstacles to mimicking the flavor and nutritional composition of traditional meat. Existing methods often result in a final cell-based meat product with a flavor, aroma, or color that is not characteristic of (or different from) slaughtered meat harvested from an animal. As an example, existing methods may include growing meat cells in a growth medium with compounds that adversely affect the flavor profile of the meat cells. The growth medium often contains aromatic amino acids that can degrade or oxidize into a complex mixture of primary and secondary compounds, including aldehydes, alcohols, and ketones. The aldehydes, ketones, and alcohols can result in undesirable aromas (e.g., "wet dog" odors) or flavors, such as rancid odors or flavors. Besides different aromas or flavors, existing methods sometimes produce cell-based meat products with a consistent, nearly homogenous pink color that lacks some of the natural red or other meat hues.

[0005] Furthermore, meat cells cultured using existing methods often lack nutritional composition compared to conventional meat, which typically has highly digestible protein with amino acids, vitamins, and minerals. Existing methods often produce cell-based meat products that lack many nutrients present in conventional meat, in part because blood does not provide nutrients. Furthermore, cell-based meat products immersed in a growth medium may also exhibit excessively high osmolality or salt-mineral composition that is not characteristic of slaughtered meat.

[0006] These, along with further issues and problems, exist with existing methods for culturing cell-based meat products. Summary of the Invention

[0007] Quick Overview The present disclosure generally describes a method of washing cells from grown cell mass to remove cell culture medium and enrich the cells with finishing medium. In particular, the disclosed method includes growing the cell mass in cell culture medium and then harvesting and washing the grown cell mass with a series of wash buffers or gradient wash buffers that change over time. One such wash buffer can replace or remove the cell culture medium. Another wash buffer can take the form of a finishing buffer that improves the cell texture and nutritional composition of the grown cell mass. Additionally or alternatively, the disclosed method can use gradient wash buffers by decreasing the concentration of the wash medium and increasing the concentration of the finishing medium over time. Whether applied separately or as a gradient mixture, the disclosed method can wash the grown cell mass with a wash medium and wash the grown cell mass with a finishing medium to remove or dilute cell culture medium residues, oxidize undesirable flavors and aromas, improve color, and / or enrich the grown cell mass with nutrients. [Brief description of the drawings]

[0008] The detailed description refers to the drawings, which are briefly described below.

[0009] [Figure 1] FIG. 1 illustrates a schematic diagram of washing and concentrating an expanded cell mass using a washing medium and a concentration medium according to one or more embodiments of the present disclosure.

[0010] [Diagram 2] FIG. 2 illustrates exemplary characteristics of the completion of a cellular threshold proliferation phase, according to one or more embodiments of the present disclosure.

[0011] [Diagram 3]FIG. 3 illustrates a multiple wash method and a gradient wash method according to one or more embodiments of the present disclosure.

[0012] [Figure 4] FIG. 4 illustrates an exemplary system including various components for washing and concentrating an expanded cell mass in accordance with one or more embodiments of the present disclosure.

[0013] [Diagram 5] FIG. 5 illustrates an exemplary wash medium composition and an exemplary concentrate medium composition according to one or more embodiments of the present disclosure.

[0014] [Figure 6] FIG. 6 illustrates flowing a first exchange medium and a second exchange medium over cells of a cell mass, according to one or more embodiments of the present disclosure.

[0015] [Figure 7] FIG. 7 illustrates a sequence of operations for washing and concentrating an expanded cell mass using a wash medium and a concentration medium according to one or more embodiments of the present disclosure.

[0016] [Figure 8] FIG. 8 illustrates a sequence of operations for washing and concentrating an expanded cell mass using a gradient wash medium in accordance with one or more embodiments of the present disclosure.

[0017] [Figure 9] FIG. 9 illustrates a series of operations for washing a cell mass with replacement medium according to one or more embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present disclosure describes one or more embodiments of a method for washing and preparing a grown cell mass to improve the texture, flavor, and aroma of a cell-based meat product. The disclosed method includes growing the cell mass and harvesting the grown cell mass after the grown cell mass completes a threshold growth stage. The grown cell mass can be harvested and separated from the cell culture medium. Whether applied in a separate wash medium or as a gradient of the wash medium, the disclosed method can wash the grown cell mass with a first wash buffer composition that replaces the residual cell culture medium, and then wash the grown cell mass with a finishing buffer containing nutrients to concentrate the grown cells.

[0019] Illustratively, the disclosed methods include growing a cell mass in a cell culture medium, removing the grown cell mass from the cell culture medium, washing the grown cell mass with a wash medium to flush away remaining cell culture medium, and washing the grown cell mass with an enrichment medium comprising nutrients preferred for human consumption. In some embodiments, rather than using a separate wash medium and a separate enrichment medium, the disclosed methods include washing the grown cell mass with a gradient wash medium by decreasing the concentration of the wash medium and increasing the concentration of the enrichment medium.

[0020] As mentioned above, the disclosed methods include growing a cell mass. The cells can be grown in a growth environment, such as a bioreactor. More specifically, suspension cells and / or adherent cells can be grown in the presence of a cell culture medium. As further described below, the cell culture medium includes an energy source and compounds for growing the cells.

[0021] After expanding the cell mass, the disclosed method further includes washing the cells when they have completed the threshold expansion phase. Generally, cells complete the threshold expansion phase when the cells slow or stop exponential growth. Cells that have completed the threshold expansion phase may have different characteristics. For example, the disclosed method may include evaluating cell density, cell metabolism, compounds present in the cell culture medium, and other factors to determine whether the cells have completed the threshold expansion phase or the threshold expansion phase and become an expanded cell mass.

[0022] As described above, the expanded cell mass can be washed with a wash medium. In some embodiments, the disclosed method includes using a series of wash media. Whether applied in separate wash media or as a gradient of wash media, the wash medium can replace or remove the cell culture medium from the expanded cell mass, for example, by flowing or spraying the wash medium onto the expanded cell mass, immersing the expanded cell mass in the wash medium, or using any other suitable washing approach.

[0023] After washing or as part of washing, the disclosed method may further agitate the grown cell mass and the washing medium. After washing the grown cell mass or as part of washing, the disclosed method may also concentrate the grown cell mass using a concentration medium. Generally, the concentration medium contains vitamins, amino acids, antioxidants, fats (e.g., oils or fat cells), and / or other compounds to improve the quality of the cell-based meat product. In some cases, the concentration medium also adds or changes the color of the grown cell mass. The concentration medium acts as a final or finishing treatment for the grown cells in the grown cell mass, increasing the nutritional value of the grown cells while balancing the ingredients in the washed grown cell mass.

[0024] When washing with a washing medium and / or an enrichment medium, the expanded cell mass can be washed with a series of media or a gradient washing medium. For example, the disclosed method can include washing the cells with a washing medium, removing the washing medium, and adding an enrichment medium. In other embodiments, the disclosed method includes washing the cells with a gradient washing medium, where the gradient washing medium flows over the cells for a set period of time, and the composition of the gradient washing medium changes over time. For example, the gradient washing medium can be applied by decreasing the concentration of the washing medium and increasing the concentration of the enrichment medium over time.

[0025] The disclosed method offers several advantages over unprocessed cell cultures or other existing and unprocessed cell-based meats. By washing the grown cell mass using a wash medium, the disclosed method removes compounds that impart flavors or aromas to the cell culture medium and cells that are not characteristic of (or distinct from) slaughtered meat. The wash medium can also adjust the ionic and pH balance within the grown cell mass to prepare the grown cell mass for enrichment media.

[0026] In addition to improving the flavor or aroma of the cell-based meat, the disclosed methods also improve the nutritional composition of the cell-based meat product compared to existing and unprocessed cell-based meats. In particular, the disclosed methods include washing the grown cell mass with an enrichment medium that includes nutrients after the cells are grown in a cell culture medium and some or all of the cell culture medium is washed away. In some embodiments, the grown cell mass takes up vitamins, amino acids, antioxidants, fats, and / or other beneficial compounds from the enrichment medium. The use of the enrichment medium improves the nutritional composition of the finished cell-based meat product.

[0027] As indicated by the preceding discussion, the present disclosure utilizes various terms to describe the features and advantages of the disclosed methods. Further details regarding the meaning of such terms are now provided. For example, as used herein, the term "cell mass" refers to a mass containing meat cells. In particular, cell mass refers to cultured meat cells that have been collected into an aggregate mass. As discussed below, the cell mass can include different cell types, such as one or more of myoblasts, mesenchymal blasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, endothelial cells, or other similar cell types. For example, the cell mass can include a cultured meat cell sheet that is grown within an enclosure, such as a chamber, housing, or container.

[0028] Relatedly, the term "expanded cell mass" refers to a cell mass that includes one or more expanded cells. For example, an expanded cell mass includes a group of cells that are nourished by a growth medium (e.g., cell culture medium) to grow during a growth period. In some cases, an expanded cell mass includes a cell mass that has completed a growth period.

[0029] As further used herein, the term "cells" refers to individual cells of meat. In particular, the cells may include different cell types, such as one or more of myoblasts, vascular endothelial cells, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, endothelial cells, or other similar cell types. Furthermore, the cells may include different types of progenitor cells, including myogenic progenitor cells, adipogenic progenitor cells, mesenchymal progenitor cells, or other types of progenitor cells.

[0030] As further used herein, the term "cell culture medium" or "growth medium" refers to a liquid or gel that contains compounds that support the growth of cells. In particular, cell culture medium contains an energy source and compounds that regulate the cell cycle. For example, cell culture medium can contain amino acids, vitamins, inorganic salts, glucose, dissolved gases, serum, growth factors, hormones, and attachment factors. Cell culture medium can also help maintain pH and osmolality during cell growth and proliferation.

[0031] As used herein, the term "washing medium" refers to a liquid for washing cells after they have grown into a proliferative cell mass. In particular, the washing medium can be utilized to wash cell culture medium from the proliferative cell mass. For example, the washing medium can include phosphate buffered saline (PBS), citric acid, citrate / dibasic potassium buffer, or other solutions for rinsing or washing the proliferative cell mass.

[0032] In contrast, the term "enriched medium" refers to a liquid for concentrating cells after they have grown into a grown cell mass. In particular, the enriched medium contains ingredients that can be added to the grown cell mass to improve the nutritional composition or otherwise improve the quality of the grown cell mass for consumption. For example, the enriched medium can contain nutrients such as vitamins, amino acids, antioxidants, fats, or other compounds important to a healthy or nutritional diet. In some cases, the enriched medium may not contain nutrients necessary for cell growth.

[0033] As used herein, the term "nutrients" refers to substances used by a cell or organism to survive, grow, or reproduce. Nutrients can also refer to substances required for an animal cell or organism to survive, grow, or reproduce. For example, nutrients can include vitamins, minerals, amino acids, antioxidants, or fats added to cells that are necessary for a cell, organism, or human to survive, grow, and reproduce.

[0034] As used herein, the term "gradient washing medium" refers to a liquid having a gradient of washing medium and enrichment medium. In particular, gradient washing medium can refer to a liquid containing a dynamic gradient that changes to decrease the concentration of washing medium and increase the concentration of enrichment medium. For example, gradient washing medium can refer to a liquid that initially contains 100% washing medium, decreases the concentration of washing medium during an intermediate time, increases the concentration of enrichment medium, and later contains 100% enrichment medium.

[0035] As used herein, the term "culture tank" refers to a vessel used to culture cells. In particular, a culture tank refers to an environment capable of holding cells and cell culture medium. For example, a culture tank can include a bioreactor system.

[0036] As further used herein, the term "separator" refers to an apparatus or method for separating materials. In particular, a separator refers to an apparatus for separating proliferated cell mass from liquids or gels, including cell culture media, wash media, enrichment media, and other media. For example, a separator can include a centrifuge, a filter, a sieve, or a flocculant used for sedimentation.

[0037] As used herein, the term "washing tank" refers to a container for washing cells. In particular, a washing tank refers to a container that holds at least one of a growing cell mass and a washing medium or a concentrated medium. For example, a washing tank can include a tank with one or more openings for adding and removing the washing or concentrated medium.

[0038] As further used herein, the term "exchange medium" refers to a liquid or gel that contains a compound that stimulates the diffusion of a substance into and / or out of a cell. In particular, the exchange medium can contain various concentrations of membrane-permeable solutes. When a cell is exposed to the exchange medium, the membrane-permeable solutes can pass through the cell membrane from an area of ​​high concentration to an area of ​​low concentration. For example, the exchange medium may contain a high concentration of nutrients that diffuse into the intracellular space of the cell. In another example, the exchange medium contains a low concentration of solutes that cause substances from within the cell to be expelled from the cell.

[0039] As further used herein, the term "hypotonic" refers to the property of a solution having a relatively low concentration of solutes compared to the concentration of solutes in another solution or cell. In particular, a hypotonic solution contains a lower concentration of membrane-permeable solutes compared to another solution. In some examples, a hypotonic solution contains a lower concentration of membrane-permeable solutes than found in the exchange medium or cell culture medium. For example, the first exchange medium can contain a hypotonic solution that stimulates membrane-permeable solutes to exit the cell.

[0040] As further used herein, the term "hypertonic" refers to the property of a solution having a relatively high concentration of solutes compared to the concentration of solutes in another solution or cell. In particular, a hypertonic solution contains a higher concentration of membrane-permeable solutes compared to a cell culture medium or exchange medium. For example, a hypertonic exchange medium can contain a relatively high concentration of membrane-permeable solutes that enter the intracellular space of cells in a cell mass.

[0041] Further details regarding the disclosed method are now provided in connection with exemplary figures depicting exemplary embodiments and implementations of the disclosed method. For example, FIG. 1 illustrates an overview of operations in the disclosed method for washing cells in a wash medium and washing cells in a concentrated medium, according to one or more embodiments. In particular, FIG. 1 shows a series of operations 100 including an operation 102 of growing cell masses, an operation 104 of removing the grown cell masses from the cell culture medium, an operation 106 of washing the grown cell masses with a wash medium, and an operation 108 of washing the grown cell masses with a concentrated medium.

[0042] As shown in FIG. 1, the sequence of operations 100 includes an operation 102 of growing a cell mass. In some cases, operation 102 includes growing cells 112 in cell culture medium 114a. For example, in some instances, cells 112 are grown in a bioreactor 110. Cells 112 may include suspension cells suspended in cell culture medium 114a. Additionally or alternatively, cells 112 may include adherent cells attached to a substrate 118. In some embodiments, operation 102 includes growing cells until the cells complete a threshold growth or threshold growth stage. FIG. 2 and corresponding paragraphs detail characteristics of cells that have completed a threshold growth stage, according to one or more embodiments.

[0043] In one example, the operation 102 of growing the cell mass includes filling the bioreactor 110 with cells and cell culture medium to 20% volume. Illustratively, if the bioreactor 110 is a 1 kiloliter tank, the operation 102 includes adding 200 L of cells and cell culture medium. The disclosed methods can include a relatively high concentration of cells (e.g., 15-30%) at the beginning of the growth period. The cells are allowed to grow and reach exponential growth. After exponential growth is achieved, the disclosed methods include filling the bioreactor 110 with cell culture medium to 100% of its volume. In other embodiments, the disclosed methods include filling the bioreactor 110 with different volumes of cells and cell culture medium.

[0044] The series of operations 100 further includes an operation 104 of removing the proliferated cell mass from the cell culture medium. In particular, operation 104 includes separating the proliferated cells 122 from the cell culture medium 114b. The proliferated cells 122 may include cells from the proliferated cell mass that are collected together or dispersed throughout the environment. As shown, the disclosed method may include utilizing a centrifuge 120 or a filter 116. For example, operation 104 may include using a centrifuge 120 to isolate the proliferated cells 122 from the cell culture medium 114b. In one or more embodiments, removing the cell mass from the cell culture medium by centrifugation is a preferred method for separating suspension cells from the cell culture medium. Suspension cells grow in solution and therefore often require more centrifugation to separate additional liquid. Thus, the disclosed method may include adding centrifugation time or cycles based on the proliferated cells being suspension cells. Similarly, samples of aggregates, or cells grown as partial sheets of tissue in suspension, often contain a higher percentage of cell culture medium compared to samples of adherent cells. Thus, the disclosed methods can include adding additional centrifugation time or cycles based on whether the cells are aggregates.

[0045] As shown, the centrifuge 120 can include a continuous centrifuge or a batch centrifuge. In continuous flow centrifugation, large volumes of material are centrifuged without the need to fill and decant multiple centrifuge tubes, as in batch centrifugation. When using a centrifuge, a mixture of growing cells and cell culture medium, in some cases, flows continuously into a rotor operating at a selected operating speed. The cells settle out of the flow, which carries the cell culture medium away from the cells. In a preferred embodiment, the centrifuge is operated at a speed that poses a low risk of cell lysis and cell death, so that the cells remain largely intact after being separated from the cell culture medium.

[0046] In contrast to continuous centrifugation, in some cases batch centrifugation may have the advantage of forming a cell pellet with a relatively high cell concentration. For example, batch centrifugation may result in nearly 100% packed cell volume with no (or little) cell culture medium remaining. However, batch centrifugation is inefficient compared to continuous centrifugation in that it may consume more time.

[0047] In some embodiments, the operation 104 of removing the proliferating cell mass from the cell culture medium can include utilizing a filter 116 to separate the proliferating cells 122 from the cell culture medium 114b. In particular, the filter 116 is a semi-permeable membrane that acts as a barrier to retain the proliferating cells 122 while allowing the cell culture medium 114b to pass through. In some embodiments, the disclosed methods use membrane filtration to separate the suspension cells from the cell culture medium.

[0048] In some embodiments, the operation 104 of removing the expanded cell mass from the cell culture medium is replaced with an operation of diluting or replacing the cell culture medium with a wash medium. For example, the expanded cell mass may simply be filled with a wash medium to dilute and / or replace the cell culture medium. This can beneficially reduce the number of separation steps (e.g., by centrifugation) by combining the separation of the cell culture medium and the wash medium into a single operation.

[0049] In addition to or as an embodiment of a filter, operation 104 can include the use of sieving and / or sedimentation. Sieving is like filtration in that cells that are too large to pass through the holes in the sieve are retained. The disclosed methods can include the use of in-line and / or shaking sieves. The disclosed methods can further include several layers of sieves to separate tissues and particulates.

[0050] In addition to or instead of filtering, operation 104 may further include flocculation or sedimentation. Flocculation or sedimentation may be a preferred method for separating aggregates or clusters of cells from the cell culture medium. In flocculation / sedimentation, a flocculant is added to collect the cells into aggregates. For example, the disclosed method may include adding a polymer, a multivalent cation, a metal salt, or other compound to aggregate the growing cells 122. After or during the aggregation of the cells, the disclosed method may drain or filter the cell culture medium.

[0051] In one or more embodiments, the disclosed methods utilize one or more of the above-described methods for removing cell clumps from the cell culture medium. For example, the disclosed methods can perform operation 104 by isolating the proliferating cells 122 from the cell culture medium using a combination of flocculation, centrifugation, and / or filtration.

[0052] In some embodiments, operation 104 is optional and the disclosed method does not include removing the cell mass from the cell culture medium. For example, in some embodiments, the grown cell mass includes tissue or cells grown as a sheet. The tissue can include adherent cells that attached to the substrate during growth. In such a case, after the growth stage, the cell culture medium is simply drained from the tank, leaving the cell tissue attached to the substrate in a relatively dry form, ready for subsequent washing. Thus, the disclosed method can proceed from operation 102 of growing the cell mass to operation 106 of washing the grown cell mass with washing medium. In such an embodiment, the disclosed method can include further agitation (e.g., mixing or inversion) of the grown cell mass and washing medium to ensure that the cell tissue does not form pockets that retain trapped cell culture medium.

[0053] 1, the series of operations 100 further includes an operation 106 of washing the expanded cell mass with a wash medium. In particular, the wash medium 124 is flowed or sprayed across the expanded cells 122. The wash medium 124 displaces or removes residual media of the cell culture medium 114b from the expanded cells 122. An exemplary composition of the wash medium 124 is described below with respect to FIG.

[0054] In some embodiments, operation 106 includes pouring wash medium 124 onto the growing cells 122 and agitating the wash medium 124 and the growing cells 122. In some examples, the wash medium 124 and the growing cells 122 are agitated by gently spinning a centrifuge or a container holding the growing cells 122 and the wash medium 124. In some embodiments, the wash medium 124 and the growing cells 122 are agitated by utilizing a mixing mechanism. Exemplary mixing mechanisms can include a motorized agitator or impeller. In some embodiments, the disclosed method includes using a cell-retaining filter to retain the growing cells 122 while they are being washed with the wash medium 124.

[0055] As discussed above, operation 106 can include a variety of wash methods. For example, operation 106 can include a multiple wash method or a gradient wash method. Figure 3 and corresponding paragraphs describe an exemplary multiple wash method and an exemplary gradient wash method according to one or more embodiments.

[0056] As further shown in FIG. 1, the series of operations 100 includes an operation 108 of washing the growing cell mass with an enrichment medium. As shown, the operation 108 includes washing the growing cells 122 with an enrichment medium 126. The enrichment medium 126 can reduce the salinity of the growing cells 122. The enrichment medium 126 can also act as a final treatment to concentrate the growing cells 122. To enrich the growing cells 122, for example, the enrichment medium 126 can include nutrients such as vitamins, amino acids, antioxidants, or fats. The nutrients improve the nutritional composition of the growing cells 122 as an edible food. An exemplary composition of the enrichment medium 126 is described below with respect to FIG. 5.

[0057] As previously discussed, the disclosed methods include washing the expanded cell mass once the threshold expansion phase is complete. Figure 2 and corresponding paragraphs describe exemplary characteristics for completion of the threshold expansion phase 202. Figure 2 illustrates determining that the threshold expansion phase is complete based on a variety of characteristics including cell density 204, packed cell volume 206, timing 208, cell metabolism 210, and protein content 212. Additionally or alternatively, in some embodiments, the disclosed methods include inducing completion 214.

[0058] As shown in Figure 2, one characteristic that indicates the completion of a threshold growth phase is cell density 204. Figure 2 shows an initial cell density 216 that corresponds to when the cells are first injected into the bioreactor. As the cells grow and proliferate, the cell density increases. For example, cells may be considered to have completed the growth phase when they reach a target cell density 218. In some embodiments, the target cell density 218 is 2 million cells / milliliter, 3 million cells / milliliter, or some density intermediate between these values. In some embodiments, the cell density 204 characteristic applies to suspension cells rather than adherent cells.

[0059] Another feature indicative of the completion of the threshold proliferation phase is the packed cell volume (PCV) 206. Generally, PCV refers to the volume of cells in a sample. FIG. 2 shows a start PCV 220 and a target PCV 222. The start PCV 220 reflects the volume of cells in a sample at the beginning of the proliferation phase. The target PCV 222 indicates that the cells will proliferate and occupy a larger volume. In some embodiments, the target PCV 222 is equal to at least 1% of the PCV. In other embodiments, the target PCV 222 is equal to any value within the range of 1%-25% PCV. In some embodiments, the packed cell volume 206 characteristic is applied to evaluate suspension cells rather than adherent cells.

[0060] In addition to or instead of the PCV, in some embodiments, the cells are considered to have completed a threshold proliferation phase based on timing 208. For example, the disclosed methods can proceed to washing the propagated cells after the cells have been propagated for a proliferation period. The proliferation period can include any amount of time. In some embodiments, for example, the proliferation period is between 6 days and 140 days.

[0061] As further illustrated by FIG. 2, cells may also exhibit completion of a threshold growth phase according to cell metabolism 210. In general, as cells grow, they collectively consume more glucose and oxygen. During exponential growth, glucose and oxygen consumption is expected to increase exponentially as well. After cells leave the threshold growth phase, glucose and oxygen consumption rise more slowly, thereby indicating that growth is slowing. Thus, in some embodiments, the methods of the present disclosure may be used to increase glucose uptake and / or O2 uptake. 2 This includes analyzing utilization to determine whether the cells have completed a threshold proliferation stage. In one example, oxygen consumption stabilization, e.g., oxygen consumption that is unchanged or minimally changing over time, indicates that the cells are ready for harvesting and therefore washing and finishing.

[0062] Further, in some embodiments, the disclosed method determines the completion of the threshold proliferation phase based on protein content 212. In general, the concentration of different proteins in the cell culture medium and cell mixture can indicate a delay in proliferation. For example, an increase in lactate dehydrogenase (LDH) content in the cell culture medium indicates cell death. In particular, LDH is a cytoplasmic enzyme that is rapidly released into the cell culture medium upon cell membrane injury. Thus, an increase in LDH content can signal the completion of the completion of the threshold proliferation phase. In another example, bicinchoninic acid (BCA) can be used to analyze the total level of protein in the cell culture medium. In particular, the BCA assay can be utilized to measure the protein content in the cell culture medium. The peak and plateau of protein levels can indicate that the cells have completed the threshold proliferation phase.

[0063] The disclosed methods can further induce completion of the threshold proliferation stage. As shown in FIG. 2, the disclosed methods can induce completion 214 by exposing the cells to an exogenous regulatory factor. The exogenous regulatory factor can cause or promote transdifferentiation of the cells to transition the cells from the threshold proliferation stage to a stage that forms mature muscle fibers, such as muscle fibers and myosin heavy chains. For example, the exogenous regulatory factor can include a transdifferentiation factor that slows cell proliferation and / or stimulates transdifferentiation.

[0064] In addition to or instead of exogenous regulatory factors, in another example, the disclosed methods include inducing completion 214 by removing the cells from the cell culture medium. Generally, when the cells are removed from the incubator, they stop or slow proliferation because they are removed from the cell culture medium that provides the nutrients necessary for continued growth.

[0065] As previously discussed, the disclosed methods include washing the expanded cell mass with a wash medium to wash away residual cell culture medium. Generally, the wash medium replaces or removes the cell culture medium from the expanded cell mass. Figure 3 and the corresponding discussion detail two different methods for washing the expanded cell mass, according to one or more embodiments. Figure 3 illustrates a multiple wash method 302 and a gradient wash method 304.

[0066] Generally, the multiple wash method 302 includes washing the grown cell mass using separate and distinct media. In general, in one example, the disclosed method includes growing cells, harvesting the grown cell mass, cooling the cells, removing the cell culture medium, and applying a first wash medium including an isotonic saline solution to provide bulk dilution of the cell culture medium and limit cell rupture from osmotic stress. The first wash medium may be centrifuged or otherwise removed from the grown cell mass. A second enrichment medium is used to remove residual wash medium while also reducing the salt concentration. The second enrichment medium may be further centrifuged or otherwise removed from the grown cell mass. As shown in FIG. 3, the multiple wash method 302 includes an operation 306 of flowing a wash medium over the grown cell mass, an operation 308 of removing the wash medium, and an operation 310 of flowing an enrichment medium over the grown cell mass. Although FIG. 3 shows one cycle of flowing a wash medium over the grown cell mass, in some embodiments the disclosed method includes several cycles of flowing a wash medium over the grown cell mass.

[0067] The multiple wash method 302 includes an operation 306 of flowing wash medium over the grown cell mass. As shown in FIG. 3, wash medium 316 is added to the grown cells 314. The grown cells 314 include concentrated cells, optionally after separation by centrifugation or filtration from the cell culture medium. In some embodiments, the grown cells 314 are washed with wash medium 316 at a ratio of cells to wash medium of 1:1 to 1:4. For example, if the grown cells 314 are 40 L in volume, the disclosed method includes adding 40 L to 160 L of wash medium 316. Further, in some embodiments, wash medium 316 is added to the grown cells 314 in a wash tank 318. The wash tank 318 may be pressurized to reduce the risk of contamination.

[0068] The amount of washing medium 316 added to the growing cells 314 can be optimized to efficiently remove remnants of cell culture medium from the growing cells 314. For example, if too much washing medium 316 is added, processing time will increase and more material will be required to process the growing cells 314. If too little washing medium 316 is used, the growing cells 314 may not be adequately covered with washing medium 316 and residual cell culture medium may remain within the growing cells 314.

[0069] The wash medium 316 may flow over or be exposed to the growth cells 314 for a wash period. The wash period can last anywhere from 5 minutes to 8 hours. If the wash period is too long and the osmolality is too high, the growth cells 314 may burst. If the wash period is too short, the wash medium 316 may not have the necessary time to equilibrate and perform the necessary ion exchange.

[0070] The disclosed methods may also include controlling the temperature of the wash medium 316. In particular, the wash medium 316 may be between -5 C and 45 C. Warmer temperatures have the advantage of faster mass transfer across the cell membrane, while lower temperatures correspond to better cell survival.

[0071] In some embodiments, operation 306 further includes agitating the mixture of washing medium 316 and growing cells 314. Generally, agitating the mixture of washing medium 316 and growing cells 314 maximizes the exposure of the growing cells 314 to the washing medium 316. The increased exposure enhances the transfer and equilibration process between the growing cells 314 and the washing medium 316. To accomplish this, the mixture may be agitated in the washing tank 318. The cells may be agitated, circulated via a pump, or homogenized using air or gas. Agitation may be limited in strength and time to preserve the cells. Excessive or overly vigorous agitation may disturb the growing cells 314 through shear forces, resulting in cell damage.

[0072] 3, the multiple wash method 302 also includes an operation 308 of removing the wash medium. In particular, the disclosed method includes separating the washed expanded cells 322 from the wash medium 316. In one or more embodiments, operation 106 utilizes the method used to separate the expanded cell mass from the cell culture medium to separate the washed expanded cells 322 from the wash medium 316. For example, operation 308 can include utilizing a centrifuge 320, filtration, sieving, or flocculation / sedimentation to separate the washed expanded cells 322 from the wash medium 316.

[0073] 3, the multiple wash method 302 includes an operation 310 of flowing an enrichment medium over the grown cell mass. In particular, the disclosed method includes flowing an enrichment medium 324 over the washed grown cells 322. The method of flowing an enrichment medium over the grown cell mass is similar to the method of flowing a wash medium over the grown cell mass. More particularly, the wash procedure, cell to enrichment medium ratio, temperature, concentration, agitation, and timing are the same or similar to those described above with respect to operation 306 of flowing a wash medium over the grown cell mass.

[0074] To illustrate, enrichment medium can be flowed over the grown cell mass and agitated. For example, the grown cell mass and enrichment medium can be agitated, circulated via a pump, or homogenized with air / gas. In some embodiments, the grown cell mass is covered with enrichment medium 324 at a ratio of cells to enrichment medium of 1:1 to 1:4. In some embodiments, the enrichment medium 324 has a temperature of 1C to 40C. More specifically, the enrichment medium 324 can be at a temperature of 4C. The disclosed methods can further include exposing the washed grown cells 322 to the enrichment medium 324 for an enrichment period. The enrichment period can be the same as the washing period, or can be longer or shorter than the washing period. In particular, the enrichment period can be from 5 minutes to 8 hours.

[0075] In some embodiments, the multiple wash method 302 further includes the additional operation of removing the enrichment medium. In particular, the enriched and washed expanded cells may be separated from the enrichment medium 324 to remove excess enrichment medium. In some examples, the disclosed method includes centrifuging the mixture of the expanded cell mass and the enrichment medium 324. Additionally or alternatively, the cells may be aggregated.

[0076] Additionally, the multiple wash method 302 can include an additional operation of drying the grown cell mass. Generally, the grown cell mass can be dried to facilitate the transition of the grown cell mass into a cell-based meat product ready for consumption, for example, by improving the texture of the cell-based meat product. For example, the grown cell mass can be dried to have a moisture content of 50%-80% to mimic the moisture content of conventional meat. For example, the grown cell mass can be dried under a vacuum, spray, or forced drying system.

[0077] Instead of the multiple wash method 302, the disclosed method can use a gradient wash method to wash the expanded cell mass. Typically, a gradient wash medium is flowed over the expanded cell mass. The composition of the gradient wash medium changes over time. For example, the wash medium can be flowed over the expanded cell mass for a set period of time, gradually transitioning to an enrichment medium.

[0078] FIG. 3 illustrates a gradient wash method 304 according to one or more embodiments. In overview, the gradient wash method 304 may include growing cells, collecting the grown cell mass, centrifuging or removing cell culture medium from the grown cell mass, cooling the cells, and washing the cells with a gradient wash medium. The gradient wash method 304 may utilize two tanks, one holding a wash medium 326 and the other holding a concentrated medium 328. The two tanks are each connected to a wash tank 332 that contains the gown cell mass 330. The concentration of the gradient wash medium may start as 100% of the wash medium 326 and slowly include more concentrated medium 328. The gradient wash medium continues to transition until it is 100% (or nearly 100%) composed of concentrated medium 328. As the gradient wash medium changes over time, the disclosed method may include draining the medium from the wash tank 332. For example, the wash tank 332 may drain the medium at the same rate that the medium is flowing into the wash tank 332. Advantages of the gradient wash method 304 over the multiple wash method 302 include improved efficiency by eliminating the need to remove wash medium.

[0079] The disclosed methods can include adjusting the time that the wash medium 326 and / or enrichment medium 328 are exposed to the gown cell mass 330. For example, as shown in FIG. 3, the gradient wash method 304 is performed within a 24 hour period. In the first 3 hours (or any time within the optimal wash period of 5 minutes to 8 hours), the gradient wash medium is characterized by an inflow of 100% (or nearly 100%) comprised of wash medium 326. Over an intermediate period from 3 hours to 21 hours, the gradient wash medium gradually transitions from an inflow having a higher concentration of wash medium 326 to an inflow having a higher concentration of enrichment medium 328. In the final 3 hours (or any time within the optimal enrichment period of 5 minutes to 8 hours), the gradient wash medium inflow is 100% (or nearly 100%) comprised of enrichment medium 328. In an alternative embodiment, the inflow transitions from less than 100% wash medium (e.g., a starting ratio of 95% wash medium and 5% enrichment medium) to less than 100% enrichment medium (e.g., a ending ratio of 5% wash medium and 95% enrichment medium).

[0080] In one example of gradient washing method 304, washing medium 326 includes a hyperosmolar solution containing high concentrations of salts, sugars, etc. Washing medium 326 can further include citrate to remove ions from gown cell mass 330. Additionally, washing medium 326 can further change the pH of gown cell mass 330. As alluded to above, in some cases, gradient washing method 304 further includes a gradual change to concentrated medium 328 with low salt concentration, little or no citrate, and additional nutrients such as vitamin B. In some embodiments, the disclosed method excludes acids (such as citrate) from washing medium 326 and concentrated medium 328 that risk protein denaturation and result in a sticky texture.

[0081] FIG. 4 illustrates an exemplary system for carrying out the disclosed methods, according to one or more embodiments. In general, FIG. 4 includes various components that may be part of a system for washing an expanded cell mass. For example, FIG. 4 illustrates a system 400 that includes a culture tank 402, a buffer tank 406, a washing tank 408, and various other components. The disclosed methods may include the use of more or fewer components than those illustrated in FIG. 4. Additionally, in some embodiments, the components of FIG. 4 are organized in a different order.

[0082] As shown in FIG. 4, the system 400 includes a culture tank 402. The culture tank 402 provides an environment in which the cells can grow and complete a threshold growth stage to become a grown cell mass. More specifically, the culture tank 402 includes a cell culture medium having nutrients for cell growth. In some embodiments, the culture tank 402 includes a bioreactor system. The cells 410 in the culture tank 402 are kept at a growth temperature. For example, the cell culture medium and cells 410 can be kept at 40° C. for mammalian cells and at a lower temperature for aquatic species. In some embodiments, the culture tank 402 also includes an agitator 412. The agitator 412 mixes or otherwise agitates the cells 410 to increase exposure of the cells 410 to the cell culture medium.

[0083] 4, the grown cell mass and cell culture medium 414 can be collected and transferred to a buffer tank 406. In some examples, a portion of the grown cell mass is collected or drained from the culture tank 402, and another portion is left in the culture tank 402 for growing the next batch. For example, 80% of the grown cell mass can be extracted from the culture tank 402 for washing, leaving 20% ​​of the grown cell mass in the culture tank 402 (e.g., for subsequent growth and propagation steps).

[0084] As mentioned above, the grown cell mass and cell culture medium 414 are transferred to the buffer tank 406 as an intermediate tank to store the grown cells during drainage and before washing. The buffer tank 406 is a sterile container that stores the cells before separation. The buffer tank 406 can provide efficiency advantages. More specifically, when several culture tanks are operating in sync, the culture tanks are drained at regular intervals. In one example with eight bioreactors operating in sync, one of them is typically drained every two hours. In some embodiments, the buffer tank 406 provides a storage space for the grown cells from the culture tanks as the downstream process catches up with the draining schedule.

[0085] As further shown in Figure 4, the system 400 further includes a heat exchanger 404a. In some embodiments, as part of washing the cells, the disclosed methods cool the grown cell mass. In the case of mammalian cells, the cells are cooled from a growth temperature of 36C-42C to 1C-20C. In some cases, the grown cell mass is cooled to the lowest temperature possible without killing or freezing the grown cells.

[0086] Cooling cells offers several advantages. For example, cooling cells slows metabolic processes within the cells, delaying lysis and other forms of cell death. Chilled cells are also less active, which extends the amount of time that further processing can take place. Additionally, cooling the expanded cell mass can increase fluid viscosity and strengthen cell membranes, allowing the expanded cells to better withstand agitation and centrifugal forces in the following steps.

[0087] Heat exchanger 404a cools the grown cell mass. In the example shown in FIG. 4, heat exchanger 404a cools both the grown cell mass and cell culture medium 414 before they are separated by separator 422. Cooling the grown cell mass and cell culture medium 414 together may strengthen the cell membranes before they are centrifuged or otherwise separated from the cell culture medium. However, cooling both the grown cell mass and cell culture medium 414 requires additional energy as opposed to cooling the smaller volume of only the grown cell mass.

[0088] In some embodiments, heat exchanger 404a cools the grown cell mass after it is separated from the cell culture medium by separator 422. In such embodiments, system 400 conserves energy because the cell culture medium is not cooled. However, because the cells in the grown cell mass are not cooled prior to separation, the cells may be more sensitive. Thus, separating the grown cell mass from the cell culture medium at growth temperatures may require centrifugation at slower speeds to prevent cell death.

[0089] As further shown in FIG. 4, separator 422 removes grown cell mass 426 from cell culture medium 424 (as described above with respect to operation 104 in FIG. 1). Separator 422 includes a continuous centrifuge that separates grown cell mass and cell culture medium 414 into a heavy phase and a light phase of centrifugation. The heavy phase contains or collects grown cell mass 426 and the light phase contains or collects cell culture medium 424. In some embodiments, the continuous centrifuge is operated at a revolutions per minute (RPM) or relative centrifugal force (G) that ensures that the integrity of the cell wall structure is maintained during centrifugation. At too high RPM or G, cells may burst. At lower RPM and G, separation is insufficient and the heavy phase contains excess growth medium.

[0090] 4, the grown cell mass 426 can be transferred to a washing tank 408 and the cell culture medium 424 can be passed through a heat exchanger 404b. The heat exchangers 404a-404b cooperate with the energy storage tank 420 to improve the energy efficiency of the system 400. Generally, the heat exchangers 404a-404b recover energy used to cool the grown cell mass.

[0091] As described above and shown in FIG. 4, heat exchanger 404a cools the growing cell mass (and in some embodiments the cell culture medium) from a growth temperature (e.g., 40 C) to a cooling temperature (e.g., 1 C). In some embodiments, the cooling temperature includes any temperature below the optimal growth temperature for the cells in the growing cell mass. Cooling the cells in heat exchanger 404a utilizes a cooled inflow 418 (e.g., 1 C) and provides a warmed outflow 416 (e.g., 36 C).

[0092] To facilitate heating and cooling, the system 400 optionally includes an energy storage tank 420. In particular, the energy storage tank 420 facilitates the efficient exchange of energy between the cell culture medium 424 at a chilled temperature and the growing cell mass at a growth temperature. More specifically, in some embodiments, the energy storage tank 420 includes a chilled liquid that sinks to the bottom and a warmed liquid that rises to the top of the energy storage tank 420. In one example, the energy storage tank 420 is a cigar-shaped energy storage tank. The warming outflow 416 warms the liquid and the cooling outflow 438 from the heat exchanger 404b cools the liquid in the culture tank 402, as shown by the different patterns of lines entering and leaving the energy storage tank 420.

[0093] As described above, the grown cell mass 426 is transferred to the washing tank 408. In the washing tank 408, the system 400 pours, sprays, or drenches the washing medium 428 and / or enrichment medium 430 onto the grown cell mass 426. The washing tank 408 may also include an agitator 440 for stirring or otherwise agitating the grown cell mass during the washing process. The agitator 440 agitates the grown cell mass and the washing medium or enrichment medium. While FIG. 4 shows an agitator 440, the cells may be agitated in the washing tank 408, circulated via a pump, or homogenized with air / gas.

[0094] 4, the disclosed methods can utilize the multiple wash methods described above in system 400. In particular, wash medium 428 can flow over the expanded cell mass in wash tank 408. The wash medium is removed from the expanded cell mass by centrifuge 432. More specifically, the wash medium can be removed using (i) a second centrifuge, or (ii) a separator 422 that is used to separate the cell culture medium 424 and the expanded cell mass 426.

[0095] The disclosed methods can also include removing the wash medium from the grown cell mass by flocculation 434. In flocculation 434, the grown cell mass is collected into aggregates by the addition of multivalent cations, metal salts, or polymers. Flocculation may be more desirable for cell-based meat products where high moisture content is tolerable. Regardless of the removal method, when the wash medium is removed, the system 400 is left with the washed cell mass 436.

[0096] As described above, when using the multiple wash method, concentrated medium is added, poured, or submerged into the washed cell mass 436. In some embodiments, concentrated medium is added to the washed cell mass 436 in the wash tank 408. In other embodiments, concentrated medium is added to the washed cell mass 436 in a separate concentrate tank.

[0097] In addition to the multiple washing method, Figure 4 also illustrates the gradient washing method described above in system 400. In particular, washing tank 408 is connected to both washing medium 428 and enrichment medium 430. As described above, the disclosed method can include flowing (or submerging) the gradient washing medium by changing the concentrations of washing medium 428 and enrichment medium 430 over time. After the gradient washing medium flows over the grown cell mass in washing tank 408, the cells can be formed into the final cell-based meat product.

[0098] As discussed above, the wash and concentrate media remove undesirable aromas, tastes, and textures while also improving the nutritional composition of the cell-based meat product. Figure 5 provides an overview of the compositions of the wash and concentrate media to provide texture, flavor, aroma, and nutritional benefits according to one or more embodiments.

[0099] As described above, the wash medium replaces / removes cell culture medium, washes the growing cell mass, and / or reduces cell lysis. The concentrated medium concentrates the growing cell mass with nutrients, preservatives (e.g., antioxidants), flavors, fragrances, colors, and / or texturizers. However, neither the wash medium nor the concentrated medium is a cell culture medium. For example, in some embodiments, neither the wash medium nor the concentrated medium contains growth factors that grow cells, growth factors that cell culture media may contain. FIG. 5 shows an example of a wash medium composition 502 and an example of a concentrated medium composition 504. In one such example of the wash medium composition 502, the wash medium is dilute phosphate buffered saline (PBS) and citric acid. The citric acid can beneficially lower cholesterol. In another example, the wash medium is a citrate / dibasic potassium buffer. The wash medium made with isotonic saline also has an osmolality that limits cell rupture, providing cost-effective bulk dilution.

[0100] In contrast, in the example of concentrated medium composition 504, the concentrated medium comprises a hypotonic saline solution with low salt content. The concentrated medium can also contain compounds that are taken up by the growing cell mass. In particular, the concentrated medium can contain proteins / amino acids, antioxidants, fats, fatty acids, oils, and vitamins. In some embodiments, the concentrated medium contains edible compounds. The concentrated medium can also contain antioxidants such as B-vitamins, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), or other natural extracts from plants with antioxidant properties such as cherry, alpha tocopherol, and other extracts. These antioxidants prevent oxidation of fats during the drying process.

[0101] Additionally, in some embodiments, the expanded cell mass is treated to improve cellular uptake of nutrients from the enrichment medium. For example, the cell mass can be heat shocked or electroporated, thereby increasing mass transfer through the cell wall. The disclosed methods can be followed by washing the expanded cell mass with enrichment medium after heat shock or electroporation.

[0102] The wash medium and / or enrichment medium can contain data that control the sensory aspects of the cell-based meat product. The sensory aspects include aroma, taste, appearance, mouthfeel, and texture. In particular, the wash medium and / or enrichment medium can facilitate the removal of volatile substances associated with inappropriate aroma. For example, many lipid oxidation products are volatile, resulting in the formation of primary and secondary compounds, including aldehydes, ketones, and alcohols, that impart inappropriate aromas and inappropriate flavors to the cell-based meat product that are not characteristic of slaughtered meat. More specifically, potentially undesirable cell culture medium components and degradants can include tyrosine, phenylalanine, tryptophan, threonine, methionine, isoleucine, choline, ammonium ions, and the like. In particular, methionine, if not degraded, can impart a stewed cabbage-like odor and flavor to the final product. Isoleucine, if not degraded, can smell and taste like cookies. Cysteine, if not degraded, can impart a rotten egg-like odor and flavor to the final product. Ammonia in the medium can impart a urine or sweat-like odor. The washing and / or enrichment medium may also remove iron and iron complexes which may be a source of off-flavors.

[0103] Furthermore, the addition of B-vitamin antioxidants in the enrichment medium minimizes the oxidative pathways that cause aroma. In particular, lipid peroxidation deteriorates traditional meat and meat products, imparting undesirable odors and textures to meat products. Typically, polyunsaturated fatty acids such as linoleic acid undergo peroxidation by reactive oxygen species (ROS). The addition of B-vitamins in the enrichment medium quenches the ROS, thereby preventing lipid peroxidation.

[0104] As mentioned above, additives in the wash medium and / or concentrate medium can also improve the taste of the cell-based meat product. In particular, the wash medium removes undesirable cell culture medium components and decomposition agents that impart off-flavors to the cell-based meat product. In one example, methionine, isoleucine, cysteine, and ammonia are all components of cell culture medium that can be washed from the grown cell mass using the wash medium. Off-flavors may be metallic in nature and can be attributed to certain species of iron present in the grown cells. Metallic components can be removed by components in the wash medium that bind the iron or replace the iron species (e.g., iron-binding proteins) with different iron species that have a more favorable flavor and / or aroma. As mentioned above, B-vitamin antioxidants in the wash medium and concentrate medium can minimize oxidation pathways that result in inappropriate flavors.

[0105] The components of the wash medium and / or the concentrate medium also improve the appearance of the final cell-based meat product. For example, color may be introduced by components with color-promoting or color-suppressing properties. Examples of such components include beet juice, carrot juice, other plant-based juices, hydrolysates or other peptones, and other colorants. Color may be retained to prevent graying by adding iron or by preventing oxidation throughout the wash and concentrate steps. Additionally, color may be removed by components with chelating properties. Examples of such components include sodium curconate, citrate salts, ethylenediaminetetraacetic acid (EDTA), and other compounds.

[0106] The wash medium and / or the enrichment medium may also include ingredients that improve the texture of the cell-based meat product. In particular, the enrichment medium may include enzymes, such as cross-linking agents, to improve the texture. For example, the enrichment medium may include transglutaminase for cross-linking. Additionally, the wash medium and the enrichment medium may include an equilibrium concentration of NaCl salt buffer, which stabilizes the proteins for an equilibrium texture.

[0107] Besides controlling the sensory aspects of the cell-based meat product, the wash medium and the concentrate medium can be used to control osmolality. Generally, cell growth media are very osmolal (e.g., cells often grow well in saline conditions), and selecting an appropriate osmolality for the wash medium and concentrate medium must be made with various considerations. If the medium has a higher osmolality than the cells, the medium components can be taken up by the cells more quickly. However, it may be necessary to first reduce the osmolality of the cells in the wash medium (e.g., to a more palatable salt content) in order to later utilize a concentrate medium that relies on high osmolality to increase cell uptake. Generally, the disclosed method may include adjusting salts (including sodium chloride (NaCl), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), and other elements) to reach a target threshold of osmolality to promote nutrient uptake.

[0108] The wash and enrichment media may also be used to alter the chemistry within the grown cell mass. In particular, the isoelectric point (pl) of conventional meat proteins tends to be around 5-5.5. The disclosed methods may include altering the pl based on pH (or vice versa) to adjust the behavior of the grown cell mass, such as ion retention and moisture content. More specifically, when the pH is lower than the pl, the proteins have an overall positive net charge, and (R-NH 3 + and R-COOH) groups are abundant. When the pH is greater than pI, the protein has an overall negative charge and (R-NH 2 When the pH is equal to pl, the proteins of the proliferating cell mass have an overall neutral charge.

[0109] Additionally, the disclosed method can include optimizing the salt concentration in the wash medium and / or concentrate medium. The salt concentration can be selected for optimal protein stability. An appropriate range of salt concentration stabilizes the protein and also contributes to a more desirable meaty texture. For example, the salt content of the medium can be altered by increasing the amount of sodium cations (Na+) and / or chloride anions (Cl-) in the wash medium and / or concentrate medium. The salt content in the wash medium and concentrate medium can be proportioned to achieve a desired ratio in the final cell-based meat product. For example, a method to reach an appropriate salt concentration includes a wash and / or concentrate medium with a concentration of Na+ or Cl- that results in a Na+ or Cl- value in the final product that is in the range of conventional meat or at least results in a more positive sensory experience. A higher wash medium / concentrate medium to cell ratio can be used to increase light exposure to maximize absorption of NaCl into the cells. Additionally, agitation can increase absorption.

[0110] Additionally, the potassium content of the wash medium and / or concentrate medium can be altered by increasing or decreasing the amount of potassium cations (K+) in the medium, similar to the method for adjusting the Na+ and Cl content in cell-based meat products. In particular, the cations in the wash medium and concentrate medium are formulated to achieve the desired ratio in the final product. Again, higher wash medium / concentrate medium to cell ratios or agitation can be used to increase light exposure to maximize absorption.

[0111] In some embodiments, the wash medium and / or enrichment medium are specific based on the cell type. For example, the medium can include chelating agents, colorants, flavorings, and antioxidants to maintain different sensory qualities corresponding to beef, chicken, duck, pork, fish, and other species. The enrichment medium can also adjust the overall macronutrient content (e.g., protein, fat, moisture, carbohydrates, etc.) and pH of the cell-based meat product to mimic the macronutrient content and pH of conventional meat.

[0112] As previously mentioned, in some cases, the disclosed methods run a series of media and buffers to improve the sensory profile and nutritional composition of the expanded cell mass. In some embodiments, the described methods are used to wash the intercellular spaces within the cell mass and add nutrients. Additionally, in some embodiments, the disclosed methods use exchange media to change the concentration of membrane-permeable solutes in the intracellular spaces within the cells of the cell mass. According to one or more embodiments, FIG. 6 and corresponding paragraphs describe utilizing exchange media to remove and / or add intracellular solutes.

[0113] In some examples, the disclosed methods include flowing an exchange medium having various concentrations of solutes and nutrients to stimulate diffusion of such solutes and nutrients into and / or out of individual cells. In diffusion, particles move from an area of ​​higher concentration to an area of ​​lower concentration until equilibrium is reached. When exchanging medium with a high particle concentration, the cells take up the particles. Exchange medium with a lower particle concentration compared to the cells causes the membrane-permeable particles to be released from the cells. By using such exchange medium, the disclosed methods can change the concentration of various solutes and nutrients in the cells. In addition to changing the concentration of particles in the cells, the exchange medium can also be used to wash particles off the surface of the cells or to attach particles to the surface of the cells.

[0114] FIG. 6 illustrates an exemplary series of exchange media according to one or more embodiments of the present disclosure. In general, cells are grown in cell culture medium. The cell culture medium is removed and a first exchange medium is added to the cells. In some examples, the first exchange medium is hypotonic with respect to the cell culture medium. More specifically, the first exchange medium contains a lower concentration of solutes compared to the cell culture medium. A second exchange medium is added to the cells. The second exchange medium may be hypertonic and may contain a higher concentration of solutes that diffuse into the cells over time.

[0115] As shown in FIG. 6, cells are grown in cell culture medium. For example, cells 602a represent adherent cells or cells grown in suspension. In either case, cells 602a grow in contact with cell culture medium 604. As previously described, cell culture medium 604 includes various solutes or components that stimulate cell growth. Some membrane-permeable solutes within cell culture medium 604 enter and equilibrate with cells 602a. As previously described, some of these membrane-permeable solutes stimulate cell growth, but also adversely affect the sensory aspects or nutrition of the cells. The disclosed method may include a step of removing undesirable membrane-permeable solutes from within cells 602a.

[0116] As further shown in FIG. 6, the disclosed method may include flushing the cells with a first exchange medium 606. In some embodiments, the first exchange medium 606 is hypotonic with respect to the cell culture medium. Thus, in some implementations, the first exchange medium includes a lower concentration of solutes compared to the cell culture medium. Over time, the membrane permeable solutes move from the intracellular space within the cell 602b to the outside of the cell 602c and reach equilibrium. As shown, the cell 602b includes a membrane permeable solute 608. In some examples, the membrane permeable solute 608 includes solutes that entered the cell by diffusion during the cell growth phase. The membrane permeable solute 608 may include undesirable concentrations of undesirable cell culture medium components or cell culture medium components, such as tyrosine, phenylalanine, tryptophan, threonine, methionine, isoleucine, choline, ammonium ions, salts, and the like.

[0117] During the wash with first exchange medium 606, at least a portion of the membrane permeable solutes 608 leave the cells to reach equilibrium with the first exchange medium 606. As shown in Figure 6, cells 602c have a lower concentration of membrane permeable solutes 608 relative to cells 602b and cell culture medium 604. In some examples, the first exchange medium 606 includes different components to reduce osmotic stress on the cells.

[0118] In some examples, the first exchange medium includes a decreasing solute gradient to reduce osmotic stress on the cells within the cell mass. More specifically, the decreasing solute gradient can begin with a solute concentration similar to that of the cell culture medium 604. The solute concentration is decreased over time to minimize osmotic shock to the cells. The solute concentration is gradually decreased until a target concentration is reached. In some embodiments, the disclosed methods include washing the cells with a series of intermediate first exchange media with decreasing solute concentrations.

[0119] As further shown in FIG. 6, the disclosed method can utilize a second exchange medium 610. In some examples, the second exchange medium is hypertonic with respect to the first exchange medium. In other examples, the second exchange medium is hypertonic with respect to the cell culture medium. In some implementations, the second exchange medium 610 includes membrane permeable solutes, including nutrients or other solutes, for diffusion into the cell. FIG. 6 shows a cell 602d exposed to the second exchange medium 610. Over time, nutrients 612 from the second exchange medium 610 enter the cell 602e by diffusion. The nutrients 612 may include membrane permeable or membrane impermeable substances. In some examples, the nutrients 612 include vitamins, amino acids, antioxidants, proteins, carbohydrates, or fats. Additionally, in some examples, at least a portion of the nutrients 612 adhere to the exterior surface of the cell 602e.

[0120] In some examples, to protect the cells in the cell mass from osmotic stress, the disclosed methods include washing the cell mass with a second exchange medium 610 by utilizing an increasing solute gradient. In particular, the second exchange medium 610 transitions from a solution having a lower concentration of the solute to a solution having a higher concentration of the solute. For example, the solution with the lower concentration of the solute can have a solute concentration similar to that of the previous solution (e.g., the first exchange medium 606 or the cell culture medium 604). A series of second exchange media with increasing solute concentrations are added up to a target second solute concentration. In some examples, the disclosed methods wash the cell mass with a series of intermediate second exchange media with progressively higher solute concentrations.

[0121] The disclosed methods may utilize different methods to determine when to stop flowing the exchange medium over the cells. For example, the disclosed methods may flow a first exchange medium (and / or a decreasing solute gradient) over the cell mass for a wash time. The first exchange medium is removed after the wash period and a second exchange medium is added to the cells. The second exchange medium (and / or an increasing solute gradient) may be flowed over the cells for the same wash time. In some examples, the second exchange medium is flowed over the cells for a different second wash period.

[0122] Additionally or alternatively, the disclosed methods include transitioning between exchange media based on effluent composition. In particular, the composition of the effluent generally indicates the solutes that have passed into or out of the cells. For example, cells initially exposed to a first exchange medium may excrete a greater amount of solutes into the first exchange medium. The effluent from this initial exposure may have a higher concentration of solutes. The first exchange medium may be flowed over the cells until the effluent composition is substantially similar to the composition of the first exchange medium. The disclosed methods may include flowing the first exchange medium until the first effluent is substantially similar to the composition of the first exchange medium. Similarly, the disclosed methods may stop the flow of the second exchange medium when the second effluent has a substantially similar composition to the second exchange medium.

[0123] In some implementations, the disclosed method includes using a single exchange medium. In particular, the exchange medium can include both a low concentration of undesirable cell-permeable solutes and a high concentration of nutrients. The low concentration of undesirable cell-permeable solutes causes the solutes to diffuse out of the cells, while the high concentration of nutrients causes the nutrients to diffuse into the cells at the same time. As with the first and second exchange medium, the disclosed method can utilize a single gradient exchange medium that slowly transitions from a composition similar to the cell culture medium to a target composition.

[0124] In some embodiments, the disclosed method includes washing the cells with an exchange medium in addition to utilizing a wash medium and a concentrated medium. For example, the disclosed method may include washing the cells with a wash medium, a concentrated medium, a first exchange medium, and a second exchange medium. In another embodiment, the wash medium and the concentrated medium include a first exchange medium and a second exchange medium, respectively. More specifically, the wash medium may include a low concentration of particulates to draw out undesirable solutes from within the cells. The concentrated medium may contain a high concentration of membrane-permeable nutrients that enter the cells.

[0125] Figures 1-6, the corresponding text, and examples provide several different systems, methods, techniques, components, and / or devices for washing and concentrating expanded cell mass, according to one or more embodiments. In addition to the above description, one or more embodiments may also be described in terms of flow charts that include operations for achieving a particular result. Figures 7-8 show flow charts of such operations. Operations described herein may be repeated or performed in parallel with each other or with different instances of the same or similar operations.

[0126] 7 shows a flow chart of a series of operations 700. Generally, the series of operations 700 includes an operation 702 of growing cell masses in a cell culture medium, an operation 704 of removing the grown cell masses from the cell culture medium, an operation 706 of washing the grown cell masses with a wash medium, and an operation 708 of washing the grown cell masses with an enrichment medium.

[0127] The series of operations 700 includes an operation 702 of growing a cell mass in a cell culture medium.

[0128] As shown in FIG. 7, the series of operations 700 includes an operation 704 of removing the expanded cell mass from a cell culture medium. In particular, operation 704 includes removing at least a portion of the expanded cell mass from the cell culture medium. In some embodiments, operation 704 further includes removing at least a portion of the expanded cell mass from the cell culture medium based on the expanded cell mass completing a threshold expansion phase. In some embodiments, the threshold expansion phase is completed when the expanded cell mass reaches at least one of a viable cell density of 3 million cells per milliliter or a packed cell volume of 1% to 25%. Additionally, in some embodiments, operation 704 further includes stimulating completion of the threshold expansion phase by exposing the expanded cell mass to an exogenous regulator.

[0129] The series of operations 700 further includes an operation 706 of washing the grown cell mass with a washing medium. In particular, operation 706 includes washing the grown cell mass with a washing medium to wash away remaining cell culture medium. In some embodiments, the washing medium does not include growth factors for stimulating the grown cell mass to grow. Furthermore, in some embodiments, operation 706 further includes washing the grown cell mass with the washing medium by agitating both the grown cell mass and the washing medium, circulating the grown cell mass and the washing medium with a pump, and homogenizing the grown cell mass and the washing medium with a gas. In some embodiments, washing the grown cell mass with the washing medium includes flowing the washing medium over the grown cell mass and flowing an intermediate medium including an increasing concentration of the enrichment medium over time. In some embodiments, washing the grown cell mass further includes flowing the washing medium over the grown cell mass and agitating both the washing medium and the grown cell mass. In some embodiments, the washing medium is at a temperature including -5C to 45C.

[0130] 7 further includes an operation 708 of washing the expanded cell mass with an enrichment medium. In particular, operation 708 includes washing the expanded cell mass with an enrichment medium that includes nutrients. In some embodiments, the enrichment medium does not include growth factors for stimulating the expanded cell mass to grow.

[0131] In some embodiments, the series of operations 700 includes the additional operation of drying the washed expanded cell mass.

[0132] 8 illustrates a sequence of operations 800. Generally, the sequence of operations 800 includes an operation 802 of growing a cell mass in a culture medium, an operation 804 of removing the grown cell mass from the culture medium, and an operation 806 of washing the grown cell mass with a gradient wash medium.

[0133] The series of operations 800 includes an operation 802 of growing cell clusters in a culture medium. In particular, operation 802 includes growing cell clusters in a cell culture medium.

[0134] 8, the series of operations 800 includes an operation 804 of removing the proliferated cell mass from the culture medium. Operation 804 includes removing the proliferated cell mass from the cell culture medium. In some embodiments, removing the proliferated cell mass from the cell culture medium includes separating the proliferated cell mass from the cell culture medium via centrifugation.

[0135] As further shown in FIG. 8, the series of operations 800 includes an operation 806 of washing the grown cell mass with a gradient wash medium. Operation 806 includes washing the grown cell mass with a gradient wash medium by decreasing the concentration of the wash medium and increasing the concentration of the enrichment medium over time. In some embodiments, the enrichment medium includes nutrients including at least one of vitamins, amino acids, antioxidants, or fats. Further, in some embodiments, the nutrients include macronutrients including at least one of proteins, fats, moisture, or carbohydrates. In some embodiments, the enrichment medium alters the pH of the grown cell mass.

[0136] In some embodiments, the series of operations 800 further includes cooling the expanded cell masses before or after removing the expanded cell masses from the cell culture medium.

[0137] 9 illustrates a sequence of operations 900 for washing cell masses with first and second exchange media, according to one or more embodiments. In particular, the sequence of operations 900 includes an operation 902 of growing cell masses in cell culture medium.

[0138] FIG. 9 further illustrates operation 904 of washing the cell mass with a first exchange medium. In some embodiments, washing the cell mass with the first exchange medium causes a first set of membrane-permeable solutes to diffuse out of the intracellular space of the cells in the cell mass. In some examples, the first exchange medium is hypotonic with respect to the cell culture medium. In some implementations, the first exchange medium has a lower concentration of membrane-permeable solutes compared to the cell culture medium. Furthermore, in some examples, the first exchange medium includes a decreasing solute gradient, where the first exchange medium transitions from a solution with a higher concentration of solutes to a solution with a lower concentration of solutes. Additionally, in some cases, flowing the first exchange medium with a decreasing solute gradient across the cell mass provides a gradual change in solute concentration, thereby reducing osmotic stress on the cells of the cell mass. In some implementations, the decrease in solute gradient begins at a solute concentration substantially similar to that of the cell culture medium.

[0139] The sequence of operations 900 includes an operation 906 of flushing the cell mass with a second exchange medium. In some embodiments, flushing the cell mass with the second exchange medium causes a second set of membrane-permeable solutes to diffuse into the intracellular space of the cells. In some embodiments, the second exchange medium is hypertonic with respect to the first exchange medium. In some instances, the second exchange medium has a higher concentration of membrane-permeable solutes compared to the first exchange medium. Additionally, the second exchange medium can include an increasing solute gradient, where the second exchange medium transitions from a solution having a lower concentration of solutes to a solution having a higher concentration of solutes. Additionally, in certain embodiments, flowing the second exchange medium including an increasing solute gradient over the cell mass provides a gradual change in solute concentration, thereby reducing osmotic stress on the cells of the cell mass. Additionally, in some instances, the increasing solute gradient starts at a solute concentration substantially similar to that of the first exchange medium. In certain embodiments, the increasing solute gradient includes an increasing concentration of nutrients. In some embodiments, at least a portion of the nutrients diffuse into the cells of the cell mass and at least a second portion of the nutrients adheres to the exterior surface of the cells.

[0140] In some embodiments, the sequence of operations 900 includes flushing the cell mass with a first exchange medium until the first effluent has a substantially similar composition to the first exchange medium. In some examples, the sequence of operations 900 includes flushing the cell mass with a second exchange medium until the second effluent has a substantially similar composition to the second exchange medium. Additionally, in some examples, the sequence of operations 900 includes washing the cell mass for a wash period.

[0141] In addition to or instead of the set of operations depicted in Figures 7-9, the disclosure includes an additional set of operations. In some embodiments, the additional set of operations includes a method for enriching a cultured meat product, the method including growing cell masses in a cell culture medium; removing the grown cell masses from the cell culture medium; and washing the grown cell masses with a gradient wash medium by decreasing the concentration of the wash medium and increasing the concentration of the enrichment medium over time. In some embodiments, the enrichment medium includes nutrients including at least one of vitamins, amino acids, antioxidants, proteins, carbohydrates, or fats.

[0142] Additionally, in addition to or in lieu of the sequence of operations depicted in Figures 7-9, the present disclosure includes an apparatus for finishing cell mass for cultured meat preparation. In some implementations, the apparatus includes a culture tank for growing cell mass in cell culture medium, a separator for removing the grown cell mass from at least one of the cell culture medium, the wash medium, or the concentrate medium, and a wash tank for storing the grown cell mass and flowing at least one of the wash medium or the concentrate medium over the grown cell mass. In some implementations, the separator includes at least one of a centrifuge or a filter. In some examples, the apparatus further includes a heat exchanger for cooling the grown cell mass below its optimal growth temperature. In some embodiments, the apparatus further includes an agitator for agitating the grown cell mass and at least one of the cell culture medium, the wash medium, or the concentrate medium.

[0143] According to common practice, various features shown in the drawings may not be drawn to scale. The illustrations presented in this disclosure are not meant to be actual diagrams of any particular apparatus (e.g., device, system, etc.) or method, but merely idealized representations used to describe various embodiments of the disclosure. Thus, dimensions of various features may be arbitrarily expanded or reduced for clarity. In addition, some of the drawings may be simplified for clarity. Thus, the drawings may not depict all of the components of a given apparatus (e.g., device) or all operations of a particular method.

[0144] The terms used in this specification, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including, but not limited to," etc.).

[0145] In addition, where a particular number of introduced claims is intended, such intent is expressly recited in the claim, and in the absence of such recitation, no such intent exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce the recitation of the claims. However, the use of such phrases should not be construed to mean that the introduction of a claim reference with the indefinite article "a" or "an" is limited to embodiments that include only one such reference, even if the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"), and the same applies to the use of definite articles used to introduce a claim reference.

[0146] In addition, even if a specific number of an introduced claim is explicitly recited, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., a plain recitation of "two enumerations" without other modifiers, meaning at least two enumerations, or more than two enumerations). Furthermore, when a convention similar to "at least one of A, B, and C, etc." or "one or more of A, B, and C, etc." is used, it is generally intended that such a configuration includes A only, B only, C only, A and B together, A and C together, B and C together, or A, B, and C together, etc. For example, use of the term "and / or" is intended to be interpreted in this manner.

[0147] Additionally, any language or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" should be understood to include the possibilities of "A" or "B" or "A and B."

[0148] However, the use of such phrases should not be construed to mean that the introduction of a claim reference with the indefinite article "a" or "an" is intended to limit any particular claim containing such a reference to embodiments containing only one such reference, even if the same claim contains the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"), and the same applies to the use of definite articles used to introduce a claim reference.

[0149] Additionally, the use of the terms "first", "second", "third", etc., is not necessarily used herein to imply a particular order or number of elements. In general, the terms "first", "second", "third", etc., are used to distinguish different elements as general identifiers. Where terms "first", "second", "third", etc., do not indicate a particular order, these terms should not be understood to imply a particular order. Additionally, where terms "first", "second", "third", etc., do not indicate a particular number of elements, these terms should not be understood to imply a particular number of elements. For example, a first widget may be described as having a first side and a second widget may be described as having a second side. Use of the term "second side" with respect to a second widget may be to distinguish such side of the second widget from the "first side" of the first widget, and does not imply that the second widget has two sides.

[0150] All examples and conditional language recited herein are intended for educational purposes to aid the reader in understanding the invention and the concepts contributed by the inventor to advance the art, and should not be construed as being limited to such specifically recited examples and conditions. Although embodiments of the present disclosure have been described in detail, it should be understood that various changes, substitutions, and alterations can be made thereto without departing from the spirit and scope of the present disclosure.

[0151] The present invention may be embodied in other specific forms without departing from the spirit and essential characteristics thereof. Indeed, the described embodiments are to be considered in all respects as illustrative only and not restrictive. For example, the methods described herein may be performed with fewer or more steps / actions, or the steps / actions may be performed in a different order. In addition, the steps / actions described herein may be repeated or performed in parallel or in parallel with each other for different instances of the same or similar steps / actions. The scope of the present invention is therefore indicated by the appended claims, rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. 1. A method for concentrating a cultured meat product, comprising: Growing the cell mass in a cell culture medium; removing at least a portion of the expanded cell mass from the cell culture medium; Washing the expanded cell mass with a wash medium to wash away residual cell culture medium; Washing the grown cell mass with a nutrient-containing enriched medium; method.

2. The proliferated cell mass is agitating both the grown cell mass and the wash medium; circulating the grown cell mass and the washing medium using a pump; and homogenizing the grown cell mass and the wash medium with a gas, thereby washing the grown cell mass with the wash medium. The method of claim 1.

3. Washing the grown cell mass with the wash medium includes flowing the wash medium over the grown cell mass and flowing an intermediate medium containing an increasing concentration of the enrichment medium over time. The method of claim 1.

4. further comprising removing at least a portion of the expanded cell mass from the cell culture medium based on the expanded cell mass completing a threshold expansion stage. The method of claim 1.

5. the threshold expansion stage is complete when the expanded cell mass reaches at least one of a viable cell density of 3 million cells per milliliter or 1% to 25% packed cell volume; The method of claim 4.

6. Washing the grown cell masses further comprises flowing the washing medium over the grown cell masses and agitating both the washing medium and the grown cell masses. The method of claim 1.

7. The wash medium is at a temperature of -5°C to 45°C. The method of claim 1.

8. 1. A method for concentrating a cultured meat product, comprising: Growing the cell mass in a cell culture medium; removing the expanded cell mass from the cell culture medium; Washing the expanded cell mass with a gradient wash medium by decreasing the concentration of the wash medium and increasing the concentration of the enrichment medium over time; method.

9. the enriched medium comprises nutrients including at least one of vitamins, amino acids, antioxidants, proteins, carbohydrates, or fats; The method of claim 8.

10. 1. A method for concentrating an edible cell-based food product, comprising: Growing the cell mass in a cell culture medium; Washing the cell mass with a first exchange medium; Washing the cell mass with a second exchange medium. method.

11. washing the cell mass with the first exchange medium causes a first set of membrane-permeable solutes to diffuse out of the intracellular spaces of cells of the cell mass; Washing the cell mass with the second exchange medium allows a second set of membrane-permeable solutes to diffuse into the intracellular space of the cells. The method of claim 10.

12. the first exchange medium is hypotonic with respect to the cell culture medium; The method of claim 10.

13. the first exchange medium comprises a decreasing solute gradient, the first exchange medium transitioning from a solution having a higher concentration of solute to a solution having a lower concentration of solute; The method of claim 12.

14. the second exchange medium is hypertonic with respect to the first exchange medium; The method of claim 10.

15. the second exchange medium comprises an increasing solute gradient, the second exchange medium transitioning from a solution having a lower concentration of solute to a solution having a higher concentration of solute; The method of claim 10.

16. the increasing solute gradient comprises increasing concentrations of nutrients; 16. The method of claim 15.

17. At least a portion of the nutrients diffuse into the cells of the cell mass; At least a second portion of the nutrients adhere to the exterior surface of the cells. The method of claim 10.

18. further comprising washing the cell mass with the first exchange medium until a first effluent has a substantially similar composition to the first exchange medium. The method of claim 10.

19. Further comprising washing the cell mass for a washing time. The method of claim 10.

20. An apparatus for finishing cell masses for cultured meat preparation, comprising: a culture tank for growing the cell mass in a cell culture medium; a separator for removing proliferated cell mass from at least one of the cell culture medium, the wash medium, or the enrichment medium; a washing tank for storing the expanded cell mass and for flowing at least one of the washing medium or the concentrated medium over the expanded cell mass; Device.

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