Bioprocessing system for de novo tissue generation and applications thereof

Abstract

Provided are systems and methods for large scale tissue culture and growth. The system may include a plurality of containers, a climate-controlled room, at least one rack having a plurality of shelves in the climate-controlled room, a movement mechanism, and at least one processor.

Description

Attorney Docket No.121430-791902 BIOPROCESSING SYSTEM FOR DE NOVO TISSUE GENERATION AND APPLICATIONS THEREOF BACKGROUND

[0001] 1. Field

[0002] The present invention relates to a bioprocessing system for de novo tissue production with applications in food, cosmetic, biotechnology and pharmaceutical industries. Use of the disclosed bioprocessing system in the field of cellular agriculture for food and other related fields is provided.

[0003] 2. Background

[0004] Bioreactors are essential tools for the culture of a variety of cells including mammalian, avian, fish, and other types of cells. Bioreactors are used in the production of biopharmaceuticals, vaccines, and other biotechnological products such as cultivated meat, proteins and complex macromolecules for food and cosmeceuticals. There are several types of bioreactors available for mammalian cell culture, including stirred-tank, rocker, air lift, and fixed-bed bioreactors. The choice of a bioreactor depends on various factors such as the scale of production, the type of cells being cultured, and the desired product.

[0005] Mammalian / avian and fish cells can be classified as either adherent or non-adherent. Adherent cells require a surface to attach to and grow, while non-adherent cells can grow in suspension. Bioreactors for adherent cells often use microcarriers to provide a large surface area for cell attachment. Non-adherent cells can be grown in suspension bioreactors without the need for an attachment to a surface.

[0006] The scale and volume of bioreactors for mammalian and animal cell culture can vary widely depending on the application. Bioreactors can range in size from small benchtop systems with working volumes of a few hundred milliliters to large-scale commercial systems with working volumes of several thousand liters. The choice of bioreactor size and volume depends on factors such as the desired yield, the type of cells being cultured, and the production process.

[0007] Typically, to exchange gas and nutrients at sufficient levels, mixing is needed. However, excessive mixing can create shear stress which can hamper the growth and function of adherent cells. Therefore, many researchers are attempting to develop non-adherent cell lines to grow them in suspension for ease of production and in higher concentrations. To do this, various techniques 1 98830719.2Attorney Docket No.121430-791902 are used such as genetic modification, long term cultures, and selection processes. However, the GMO process for food and other applications production may not be widely acceptable and may be challenged by consumers.

[0008] Growing adherent cells is typically done on a surface such as a cell culture flask in a static condition, a wall of rotating bottles, or on a surface of a microcarrier in a stirred bioreactor or on scaffolding materials. Many animal cells other than cells that are exposed to blood flow are growing and proliferating without flow shear stress in their native environment. Thus, current practices for large scale production of adherent cells, where the cell are exposed to significant shear stress in bioreactors are not optimal for large scale production of tissues, proteins deposits, or biomass. In contrast to many fermentation and bioprocessing systems, in which the target is production of a soluble protein for further harvest, via purification methods, animal tissues such as muscle tissue (meat) is a mass of cells and extracellular proteins that need to be deposited by cells for further harvest. After harvest of tissues, no further processing typically takes place for purification of harvested tissue. The extracellular protein deposited by cells is of significance, however, since culturing cells in suspension prevents the assembly of extracellular matrices and may hamper the hypertrophic cellular growth in which the biomass increases via cell doubling, rather than increasing the volume of cells.

[0009] Large scale stirred bioreactors are large stainless steel tanks equipped with sophisticated controllers to adjust parameters of the bioreactor and culture media. These systems are connected with several tubes to axillary liquids and gas tanks, creating a complex network of pipelines. The complex networks of pipelines create significant challenges when attempting to clean and sterilize the bioreactor in place. Typically, cleaning and sterilization requires high pressure steam which is energy intensive and provides many safety challenges. Due to the requirement of high pressure steam, the bioreactors need to be built with high pressure standards, increasing the cost of large scale production equipment that is not directly essential for a cell culture process.

[0010] Therefore, there is an unmet need in the field for a bioprocessing system that is mobile, low maintenance, and efficient. SUMMARY OF THE INVENTION

[0011] According to various aspects, the present disclosure relates to a method for large scale agriculture. The method includes loading a plurality of cells and a culture media into a plurality of containers at a depth of about 0.1 cm to about 100 cm; placing the plurality of containers containing the plurality of cells and the growth media on one or more shelves of at least one rack 2 98830719.2Attorney Docket No.121430-791902 in a climate-controlled room; receiving, via a processor, data from the one or more sensors; determining, via the processor, that the plurality of cells in a container has grown to a plurality of tissues ready for harvest; moving the container with the plurality of cells and / or tissues ready for harvesting to a harvesting location; and harvesting the plurality of cells and / or tissues from the container with the plurality of cells and / or tissues ready for harvesting. In an aspect, each container may include a bottom surface, one or more side surfaces extending from and surrounding the bottom surface, a top opening, a lockable lid, and one or more sensors. In an aspect, the plurality of tissues may include extracellular proteins. In another aspect, each container may have an outer bottom surface operable to stack to the lockable lid of another container.

[0012] In an aspect, the plurality of cells may be cultured in a static condition. In another aspect, the method may further include providing motion to each container about every 30 minutes to about every hour via a movement mechanism. The movement mechanism may provide angular change, rotational movement, or vibration to each container. In another aspect, the method may further include mixing the plurality of cells with a plurality of microcarriers prior to loading the plurality of cells into the plurality of container and loading a plurality of bare microcarriers into the plurality of containers prior to moving each container to the climate-controlled room. The plurality of bare microcarriers may allow immigration of the cells from the mixed cells and microcarriers to the plurality of bare microcarriers. In an aspect, the plurality of cells mixed with microcarriers are about 2% to about 60% v / v.

[0013] In another aspect, the bottom surface may be patterned with grooves to increase surface area and / or induce cell alignment and / or accelerate the tissue growth. In an aspect of the invention, the grooves possess different cross-sectional profile, such as triangle, square, sinusoid, semi-circle, others, or a combination thereof, to increase the cell and extracellular matrix deposition. In one aspect, the amplitude of profile is 0.5 to 5 millimeters and preferably 1 to 3 millimeters. In another aspect of invention, the wavelength of profile is 0.5 to 5 millimeters and preferably 1 to 3 millimeters. In an aspect, the bottom surface may be patterned with vertical posts to support three-dimensional tissue formation. In an aspect, the bottom surface has a rough surface to increase surface area and harvestable yield of cells and tissues.

[0014] In various aspects, the method may further include applying a first coating to the bottom surface of each container prior to loading the plurality of cells into each container. The first coating may be cell(s) and / or protein(s) non-adhesive. In another aspect, the method may further include applying a second coating on top of the first coating. The second coating may be configured to 3 98830719.2Attorney Docket No.121430-791902 preserve extracellular matrix (ECM) proteins. In another aspect, the method may further include applying a second coating on top of the first coating. The second coating may be cell adhesive proteins. The cell adhesive proteins may be one or more of gelatin, collagens, fibronectin, elastin, vitronectin, or other proteins from natural or recombinant sources.

[0015] In another aspect, the method may further include applying a first coating to the bottom surface of each container prior to loading the cells into the container. The first coating may be cell adhesive proteins. The cell adhesive proteins may be one or more gelatin, collagens, fibronectin, elastin, vitronectin, or other proteins from natural or recombinant sources. In one aspect of invention, the adhesive protein such as collagen are extracted from the egg-shell membrane. In another aspect, the method may further include applying a first coating to the bottom surface of each container prior to loading the cells into each container. The first coating may be cell adhesive proteins extracted from a prior cell culture of the same species. In another aspect, the method may further include applying a second coating on top of the first coating. The second coating may be cell adhesive proteins extracted from a prior cell culture of the same species.

[0016] In an aspect, the method may include functionalizing the first and / or second coating to guide the cells and provide preferential tissue formation. The first and / or second coating may be functionalized using a laser, microcontact printing, molding, stamping, chemical reactions, or combinations thereof. In another aspect, the method may further include moving each container to a sterilization location prior to loading each container with plurality of cells and / or after harvesting the plurality of cells and / or tissues from each container and sterilizing each container. In an aspect, sterilizing each container may include one or more of dry heat in an oven, high pressure steam, hydrogen peroxide fogging, ethylene oxide, irradiation, or combinations thereof.

[0017] In an aspect, the climate-controlled room may have a temperature of about 15 degrees Celsius (C) to about 40 degrees C, a humidity level of at least 80%, and an atmosphere of about 85% to about 97% oxygen and about 3% to about 15% CO2. In an aspect, the harvested cells and / or tissues are muscle tissue, fat tissue, connective tissue or combination thereof that is equivalent to meat products. In an aspect, moving each tray between the sterilization location, climate-controlled room, and / or harvesting location is conducted by an automated robotic system, automated machinery, conveyer system or manually by a human. In another aspect, the processor controls the automated robotic system and causes the automated robotic system to move each container between the sterilization location, climate-controlled room, and / or harvesting location based on data from the one or more sensors. 4 98830719.2Attorney Docket No.121430-791902

[0018] Further provided herein is a system for large scale tissue production. The system may include a plurality of containers, a climate-controlled room, at least one rack having a plurality of shelves in the climate-controlled room, a movement mechanism operable to provide a force to each container, and at least one processor. Each container may include a bottom surface, one or more side surfaces extending from and surrounding the bottom surface, a top opening, one or more sensors, and a lockable lid. Each shelf may be operable to hold at least one container of the plurality of containers. The at least one processor may be operable to receive data from the one or more sensors, determine a status of each container, and cause motion of each container every 30 minutes to about every hour via the movement mechanism. Each container may have a total volume of about 1 L to about 1000 L. Each container may be configured to contain a plurality of cells in a growth media.

[0019] In another aspect, the movement mechanism may provide angular change, rotational movement, or vibration to each container. In an aspect, each container is made of or coated with polymers, hydrogels, glass, ceramics, metals, or combinations thereof. In an aspect, each container is made of stainless steel. In an aspect, the bottom surface has a first coating that is cell(s) and protein(s) non-adherent. In another aspect, the bottom surface has a second coating on top of the first coating configured to preserve ECM proteins.

[0020] In various aspects, the lockable lid has a filter screen having a plurality of pores with a diameter of less than 0.2 µm made from a range of materials including polymer, glass, ceramics, metals, or combinations thereof, or a gas permeable silicon membrane. In an aspect, the lockable lid has an opening to an external atmosphere for gas exchange and the opening is configured to prevent entrance of free floating particles into the container. In an aspect, the climate-controlled room has a temperature of about 15 degrees C to about 40 degrees C, a humidity level of at least 80%, and an atmosphere of about 85% to about 97% oxygen and about 3% to about 15% CO2. In an aspect, each container is operable to contain a plurality of microcarriers carrying the plurality of cells and a plurality of bare microcarriers. In an aspect, the cells may immigrate between the microcarriers carrying cells and the bare microcarriers.

[0021] In another aspect, the one or more sensors are glucose, lactate, pH, dissolved oxygen, temperature, CO2, and / or volatile organic gas sensors. In another aspect, the bottom surface of each container is patterned with grooves to increase surface area and / or induce cell alignment and / or accelerate the tissue growth. In an aspect, the system may further include an automated robotic movement system operable to move each container between a cell loading location, the climate-controlled room, a harvesting location, a sterilization location, and a cleaning location. In 5 98830719.2Attorney Docket No.121430-791902 another aspect, the at least one processor is configured to control movement of each container by the automated robotic movement system based on the data received from the one or more sensors and the status of each container. In an aspect, the automated robotic movement system reduces the risk of contamination by at least 50% or more. In another aspect, the one or more sensors are operable to detect a contamination of each container before moving each container to the harvesting location. When a container is contaminated, the container is excluded from the harvesting location. In an aspect, the status of each container is one of loading, culturing, ready for harvest, harvesting, sterilizing, cleaning, coating, or annealing.

[0022] Further provided herein is a method of processing a container. The method may include cleaning and rinsing the container, drying the container, sterilizing the container, applying a first coating to the container, annealing the first coating onto the container, applying a second coating to the container, annealing the second coating onto the container, loading the container with a plurality of cells and a growth media, culturing the plurality of cells in the container in the climate- controlled room, harvesting a plurality of tissues and extracellular proteins grown form the plurality of cells, and repeating the method steps a desired number of times. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The description will be more fully understood with reference to the following figures, which are presented as variations of the disclosure and should not be construed as a complete recitation of the scope of the disclosure, wherein:

[0024] FIGS. 1A is an example of containers made of stainless steel with a lid made of gas permeable silicone rubber. FIGS.1B is an example of containers made of stainless steel with a stainless steel lid.

[0025] FIG.2 is a large scale cellular agriculture system in one example.

[0026] FIG.3 is a large scale cellular agriculture system in one example.

[0027] FIG.4 is a large scale cellular agriculture system in one example.

[0028] FIG.5A is a side view of a container in one example.

[0029] FIG.5B is an isometric view of a container in one example.

[0030] FIG.5C is a side view of a plurality of containers stacked in one example.

[0031] FIG.5D is a side view of a bottom surface of one container stacked on top of a lockable lid of another container. 6 98830719.2Attorney Docket No.121430-791902

[0032] FIG.5E is an isometric view of a plurality of containers stacked in one example.

[0033] FIG.6 is a flowchart of a method for large scale cellular agriculture in one example.

[0034] FIG.7A is a top view of a bottom surface of a container in one example.

[0035] FIG. 7B is a top view of a container with a plurality of bottom surfaces (trays) in one example.

[0036] FIG.7C is a side view of a container with a plurality of bottom surfaces (trays) in one example.

[0037] FIG.8 is a flowchart for loading a container having a plurality of bottom surfaces (trays) in one example.

[0038] FIG.9 is a diagram of various locations in a large scale cellular agriculture system in one example. DETAILED DESCRIPTION

[0039] The following detailed description illustrates various aspects of the present inventive concept. The description is intended to describe aspects of the present inventive concept in sufficient detail to enable those skilled in the art to practice the present inventive concept. Other components can be utilized, and changes can be made without departing from the scope of the present inventive concept. The following description is, therefore, not to be taken in a limiting sense. The scope of the present inventive concept is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0040] The present disclosure is based, in part, on the discovery that lower maintenance bioprocessing systems have more efficient outcomes than the high maintenance systems currently known in the art. For example, the bioprocessing system provided herein may have the same or greater efficiency in tissue production. Small production sizes increase efficiency and ease of operation. The system provided herein is capable of outputting similar or greater amounts of tissue or biomass than bioreactors and other systems known in the art, while maintaining greater ease of operation. The present disclosure is also based on the discovery that the cells used in disclosed tissue production system maintain their ability to produce and assemble extracellular matrix proteins as compared to cells in suspension systems or highly agitated culturing methods, thereby producing a higher relative yield of deposited biomass per volume of culturing media. Further, the bioprocessing systems described herein have less risk of 7 98830719.2Attorney Docket No.121430-791902 contamination. The bioprocessing systems and methods described herein are operable to grow cells and / tissue products. I. System

[0041] In some aspects, the current disclosure encompasses a bioprocessing system for large scale cellular agriculture. As used herein, the term “cellular agriculture” refers to the field of growing animal tissue products directly from cell cultures instead of using livestock. As illustrated in FIGS. 2-4, the bioprocessing system 200 may include a plurality of containers, a climate- controlled room, at least one rack 300 having a plurality of shelves 302, a movement mechanism, and at least one processor.

[0042] FIG.9 illustrates various locations that may be included in the system 200. For example, the system 200 may include a cleaning location 900, a drying location 902, a sterilization location 904, a coating location 906, an annealing location 908, a loading location 910, a climate-controlled room 912, and a harvesting location 914. The locations may all have various subsystems which are described in further detail herein.

[0043] FIGS.1A-B and 5A-E illustrate a container 100 in one example. The container 100 may include a bottom surface 502, one or more side surfaces 102 extending from and surrounding the bottom surface 502, a top opening, one or more sensors, and a lid or a lockable lid 104. In some examples, the container 100 may be made of polymers, hydrogels, glass, ceramics, metals, or combinations thereof. In an example, the container 100 may be made of stainless steel. In a further example, the container 100 may be coated with polymers, hydrogels, glass, ceramics, metals, or combinations thereof. In some examples, the container 100 may have a complex air path design to minimize the entrance of free floating particles.

[0044] The container 100 may be operable to contain a plurality of cells in a culture media to produce tissue or meat products. In some examples, a plurality of microcarriers may be loaded with the plurality of cells and carry the plurality of cells. In some examples, the cells and culture media may be mixed with microcarriers at about 2% to about 60%, about 5% to about 30%, or about 20% v / v. In some examples, bare microcarriers may be loaded with the plurality of microcarriers carrying the cells and culture media. In some examples, the bare microcarriers may be operable to allow cell migration between the microcarriers carrying cells and the bare microcarriers. Using bare microcarriers may support cell passaging without the need for cell detachment procedures using trypsin or other procedures known in the art. In some examples, 8 98830719.2Attorney Docket No.121430-791902 the containers 100 may be loaded at a depth of about 0.1 cm to about 5 cm. In another example, the containers may be loaded at a depth of about 0.1 cm to about 100 cm.

[0045] In some aspects, the container 100 may be smaller than commercially available bioreactors. The container may have a volume of about 0.5 liters (L) to about 1000 L. In another example, the container may have a volume of about 0.5 L to about 5 L, about 5 L to about 50 L, about 50 L to about 100 L, about 100 L to about 150 L, about 150 L to about 200 L, about 200 L to about 250 L, about 250 L to about 300 L, about 300 L to about 350 L, about 350 L to about 400 L, about 400 L to about 450 L, about 450 L to about 500 L, about 500 L to about 550 L, about 550 L to about 600 L, about 600 L to about 650 L, about 650 L to about 700 L, about 700 L to about 750 L, about 750 L to about 800 L, about 800 L to about 850 L, about 850 L to about 900 L, about 900 L to about 950 L, about 950 L to about 1000 L, or more. In another example, the container may have a volume of less than 0.5 L.

[0046] As illustrated in FIGS.5A-E, the outer surface of the bottom of each container may be operable to stack on top of the lockable lid of another container. In this manner, multiple containers may be stacked on top of one another.

[0047] As illustrated in FIGS. 7A-C and 8, each container may have a plurality of bottom surfaces (e.g., trays) operable to stack on top of one another. Each bottom surface may have side walls surrounding and extending from the bottom surface such that a plurality of cells and culture media may be loaded into each of the bottom surfaces. The plurality of bottom surfaces may allow the container to have multiple stages (e.g., multiple bottom surfaces / trays) thereby increasing the surface area to grow the tissue in each container. In some examples, the container may be a tote having multiple internal bottom surfaces (e.g., trays). In this example, the lockable lid may have one or more venting lines, a gas inlet port, and a gas outlet port. FIG.8 illustrates an example loading process for the container have a plurality of bottom surfaces (e.g., trays). The container and all bottom surfaces (e.g., trays) may first be sterilized. Then the plurality of cells and culture (growth) media may be loaded into each bottom surface (e.g., tray). Each bottom surface (e.g., tray) may be slid into the container. The container may have a plurality of internal sets of projections. Each projection in each set may align with another corresponding projection on an opposite side of the container. The sets of projections may each be operable to hold one bottom surface / tray. The lockable lid for the container may then be locked (e.g., closed). The container may then be rotated.

[0048] In some aspects, the bottom surface 502 of the container 100 may be patterned to increase the surface area of the bottom surface and / or induce cell alignment and / or to accelerate 9 98830719.2Attorney Docket No.121430-791902 tissue growth. For example, the bottom surface 502 may have one or more grooves. The cross- sectional groove profile may be triangle, square, sinusoid, semi-circle, any other geometries or a combination thereof. In another example, the bottom surface 502 may be patterned by any geometrical void, wells, and / or meshwork. Such grooves, wells and / or meshwork are filled with tissue during the de novo tissue generation similar to the wound healing process. This mechanism particularly is of importance for the development of the contractile tissues such as muscle and connective tissues, where accumulated contractile forces in growing tissues on flat or convex surfaces, result in detachment and loss of grown tissues. In contrast, as detailed in the disclosed invention, voids, wells, meshwork, and / or grooves on surface, not only prevent detachment, but also accelerate the tissue formation to fill the grooves and voids by accelerating the tissue regeneration at the surface of concave tissues. In another example, the bottom surface 502 may be patterned with vertical posts to support three-dimensional tissue formation. In another example, the bottom surface 502 may have a rough surface area in any geometry to increase the surface area of the bottom surface. In further examples, the bottom surface 502 may be patterned to have any type of geometry (e.g., projections or indents) to promote cell growth and increase surface area.

[0049] In another aspect, the bottom surface 502 may have one or more coatings. In some examples, a coating may be a cell and protein non-adhesive coating. In some examples, a coating may be cell adhesive proteins such as gelatin, collagens, fibronectin, elastin, vitronectin, other proteins from natural or recombinant sources, or combinations thereof. In an example, a coating may be a coating configured to preserve extracellular matrix (ECM) proteins. The ECM preserving coating may be formulated to improve adipogenesis or myofibrogenesis.

[0050] In another example, a coating may be food grade silicon functionalized with cell adhesive molecules. In some examples, the food grade silicone surface may be a smooth surface. The smooth silicone surface may be less adherent to cells, therefore after cell confluency and formation of contractile force in tissue, the tissue may detach from the surface spontaneously and facilitate the harvest of the cultured cells / tissue. A coating may be functionalized polyethylene glycol (PEG). The PEG surface may be protein and cell non-adhesive. The PEG surface may be functionalized with ECM proteins using chemical reactions. By adjusting the functional protein density, the PEG surface may support cell adhesion temporarily, similar to the silicone surface, and then release the contractile tissue for further harvest.

[0051] In some aspects, a coating may be egg white. The egg white may provide an intermediate layer that, after proliferation, improves the detachment of tissue from the bottom 10 98830719.2Attorney Docket No.121430-791902 surface of the container without using enzymatic methods such as trypsin, which destroys the ECM proteins and reduces production yield.

[0052] In some aspects, a coating may be intra and extracellular proteins extracted from a previous cell culture of the same species. The intra and extracellular proteins may be harvested from the previous cell culture, diluted, and applied as a coating for a future cell culture to improve cell adhesion and proliferation. In this particular example, there is no need to outsource coating proteins. The production line can internally source such proteins and avoid the entrance of exogenic protein or material. In some aspects, the extracted collagen protein from egg-shell membranes may use to improve the cell adhesion. Egg shell protein is a byproduct of the egg industry and is available at economic prices and massive scale, thus providing a sustainable alternative to other exogenic proteins.

[0053] In some aspects, other types of coatings may be used to facilitate tissue harvest. For example, pH, light, and heat responsive materials may be used as a coating to facilitate harvest. In another example, any type of coating that is edible and non-toxic may be used.

[0054] It will be appreciated that the coatings described herein may be combined and / or layered on top of each other. For example, the bottom surface 502 may have two coatings with a first coating being cell and protein non-adhesive while the second coating is a protein coating. Any combination of the coatings described herein may be used depending on the particular type of cellular agriculture to be conducted.

[0055] In some aspects, the one or more coatings may be annealed on to the bottom surface 502. In some examples, the system may include an annealing system in an annealing location 908 that provides the correct heat and / or pressure and / or humidity. The annealing system may be an oven or continuous conveyor oven. The annealing temperature may be about 35 degrees C to about 100 degrees C. The pressure provided to the annealing system may be atmospheric pressure. The annealing system may also provide a desired humidity. In some examples, the desired humidity may be about 0% humidity to about 100% humidity including any range or value therebetween.

[0056] In some aspects, the one or more coatings may be etched or patterned. For example, the coatings may match the pattern of the bottom surface or may be etched or patterned in a different manner. In some examples, the coatings may be etched or patterned using a laser, microcontact printing, molding, stamping, or other etching / patterning techniques known in the art. In some examples, the patterned surface may serve as a guide for cells and preferential tissue 11 98830719.2Attorney Docket No.121430-791902 formation. In an example, a parallel patterned coating may be used for alignment of the cells, such as muscle precursor cells, and yield higher muscle fiber formation. Such aligned myofibers may further improve the texture of harvested tissue to better resemble native meat fibrillar structure. Other pattern types may be used to promote other types of tissue growth.

[0057] In some aspects, the container 100 may include one or more side surfaces 102. In some examples, the container may be square, rectangular, circular, any other shape operable to promote cell growth and tissue formation, or any combinations thereof. In some examples, all or part of the interior of the side surfaces 102 may be coated using the coatings described herein.

[0058] In some aspects, the container 100 may include a top opening. In some examples, the top opening may further include a filter screen with pores having a diameter of less than 1 µm and preferably about 0.2 µm. The filter screen may be operable to promote gas exchange but also prevent the introduction of floating particles and / or microorganisms. In some examples, the filter screen may be made of natural or synthetic polymers, metals and ceramics. In some examples, the filter screen may be operable to screw on to the one or more sides at the top opening. In another example, the filter screen may be a snap fit or use other attachment mechanisms such as magnet to attach to the one or more sides at the top opening. In another example, a gas permeable membrane may cover the top opening for efficient gas exchange and to prevent the introduction of floating particles or microorganisms. In an example, the gas permeable membrane may be a silicone membrane. In some examples, the gas permeable membrane may be attached to the top opening at the one or more sides using attachment mechanism known in the art.

[0059] In some aspects, the container 100 may include a lockable lid 104. In some examples, the filter screen and / or gas permeable membrane described herein may be integrated into the lockable lid 104. In another example, the filter screen and / or gas permeable membrane may be a separate component from the lockable lid 104. In some examples, the lockable lid 104 may be operable to completely enclose the container (e.g., restricting any gas exchange). In another example, the lockable lid 104 may have an opening operable to promote gas exchange with the external atmosphere. In this example, the opening on the lockable lid 104 may be configured such that free floating particles are blocked from entering into the container 100 (e.g., using a gas permeable membrane or filter screen on the opening of the lockable lid).

[0060] In some examples, the lockable lid may have a gas inlet port and a gas outlet port in fluid communication with a gas source and a ventilation system, as illustrated, for example, in FIGS.7C and 8. The gas source may contain N2, oxygen, and CO2. In some examples, the gas source may be pressurized gas container. In some examples, the gas may be a humid gas. In 12 98830719.2Attorney Docket No.121430-791902 some aspects, the humid gas may have a humidity of at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In some examples, the humid gas may comprise N2(nitrogen), O2(oxygen), and CO2(carbon dioxide). In another example, the humid gas may comprise oxygen and CO2. The humid gas may be about 85% to about 97% oxygen and about 3% to about 15% CO2.The pressurized gas container may have a valve that is actuated allowing gas to flow through am inlet tube and into the inlet port of the lockable lid for each container.

[0061] In some aspects, the container 100 may include one or more sensors. In some examples, the one or more sensors may be a glucose, lactate, pH, dissolved oxygen, temperature, CO2, or volatile organic gas sensor. In another example, the one or more sensors may further include an identification sensor, location sensor, a movement sensor, a pressure sensor, a peroxide sensor, a volume sensor, an electrochemical sensor, an electrical sensor, or other sensors. The electrical sensor may be operable to send an electrical signal through the plurality of cells and determine a resistance, impedance, or conductivity to determine an amount of cell adhesion and / or tissue growth and / or deposited protein on the bottom surface. The electrical sensor may be a resistance, impedance, or conductivity sensor, or combinations thereof. In some examples, the one or more sensors may be in communication with at least one processor. The one or more sensors may be operable to send signals (e.g., readouts) from the one or more sensors to the at least one processor. In an example, the one or more sensors may provide real time data to the at least one processor.

[0062] In some aspects, the system 200 may further include a climate-controlled room. The climate-controlled room may be any kind of climate-controlled environment including, but not limited to, a warehouse, any kind of space, a cabinet, or any other area where the climate is controlled. In an example, the climate-controlled room may be referred to as an incubator. In some examples, the climate-controlled room may be the container itself. The climate-controlled room may be operable to have a temperature of about 15 degrees Celsius (C) to about 40 degrees C. In some examples, the temperature in the climate-controlled room may depend on the type of cells and tissues to be grown. For example, the temperature may be about 37 degrees C for non- primate animal cells, about 38 degrees C for avian cells, about 15-30 degrees C for fish cells, about 20 degrees C for shrimp cells, and about 20-36 degrees C for reptiles. The climate- controlled room may be operable to have an atmosphere of about 85% to about 97% oxygen. In an example, the climate-controlled room may be operable to have an atmosphere of about 3% to about 15% CO2. In some examples, the climate-controlled room may be operable to regulate the humidity in the climate-controlled room. For example, the climate-controlled room may be 13 98830719.2Attorney Docket No.121430-791902 operable to maintain a humidity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more. In some examples, the humidity is above 90% to avoid water loss in the culture media.

[0063] In some aspects, the climate-controlled room may have at least one rack 300, as illustrated, for example, in FIGS.2-4. In some examples, the climate-controlled room may have two racks to ten thousand racks. Each rack may have one or more shelves 302. In some examples, each rack 300 may have two, three, four, five, six, seven, eight, nine, ten, or more shelves 302. In an example, each rack 300 may have two shelves 302 to ten thousand shelves 302. In an example, each shelf 302 may be sized to hold at least one container 100.

[0064] In some aspects, the container 100 loaded with the plurality of cells and the culture media may remain on the shelf 302 in the climate-controlled room for a period of time sufficient for the plurality of cells to grow into a plurality of tissues and deposited extracellular proteins. The container 100 may be ready to be harvested when the plurality of cells has grown into a sufficient plurality of tissues and deposited extracellular proteins.

[0065] In some aspects, the system 200 may further include a movement mechanism. The movement mechanism may be operable to provide a force to each container 100. The movement mechanism may be operable to provide non-frequent short time movement. For example, the movement mechanism may be operable to provide movement to each container 100 every 30 minutes to every hour, every hour to every two hours, every two hours to every three hours, every three hours to every four hours, or more. In some examples, the movement may comprise angular change, rotational movement, and / or vibration. For example, the movement mechanism may tilt each container 100 for a period of time and then return each container 100 to its original position. In some examples, the system 200 may include a plurality of movement mechanisms. In an example, each shelf 302 in the system 200 may have its own movement mechanism. In some examples, the movement mechanism may be an agitation plate (e.g., for providing vibrational movement), a rotational plate (e.g., for providing rotational movement), or a tilting plate (e.g., for providing angular change). In some examples, the motion provided by the movement mechanism may promote cell immigration and growth without providing unnecessary shear stress.

[0066] In some aspects, the system 200 may include a robotic (e.g., automated) movement system. The robotic movement system may include various robotic components to move the containers throughout different locations in the system. For example, the system 200 may include a cell loading location 910, the climate-controlled room 912, a harvesting location 914, a cleaning location 900, a sterilization location 904, a coating location 906, and an annealing location 908. 14 98830719.2Attorney Docket No.121430-791902 In some examples, all or some of the locations in the system 200 may be located in the climate- controlled room. In other examples, the cell loading location 910, harvesting location 914, cleaning location 900, sterilization location 904, coating location 906, and annealing location 908 may be located in a different area of the system 200 away from the climate-controlled room 912. In an example, the robotic system may include automated rollers, conveyor belts, automated vehicles, robotic arms, and other automated apparatuses to move each container 100 throughout the locations in the system 200. In one aspect, the containers may move through the climate- controlled room on conveyer belts, to enhance the productivity via continuous production processes. Thereby the conveyer length, speed accommodates the necessary residence time for specified tissue growth.

[0067] In some aspects, the robotic movement system may reduce the risk of contamination to the components of the system. The robotic movement system may reduce the risk of contamination by at least 10% to at least 20%, at least 20% to at least 30%, at least 30% to at least 40%, at least 40% to at least 50%, at least 50% to at least 60%, at least 60% to at least 70%, at least 70% to at least 80%, at least 80% to at least 90%, or at least 90% to at least 99%.

[0068] In some aspects, the system 200 may include a cleaning location 900. The cleaning location 900 may have a cleaning system operable to provide a cleaning solution to the containers when cleaning is desired. After each container 100 has been sprayed with the cleaning solution, the cleaning system may rinse each container using water. Each container 100 may then be allowed to dry for a period of time. In some examples, the cleaning system may be a series of pumps and hoses operable to spray each container. In other examples, the cleaning system may include nozzles to provide pressurized cleaning solutions and water to each container. In an example, the cleaning system may be any system operable to spray or clean the containers. The cleaning system may further include rotation mechanisms and other movement mechanisms to orient each container 100 in desired directions to ensure the entire container 100 is cleaned. After the containers 100 are rinsed, they may be allowed to dry for a period of time. In some examples, the cleaning system may include a pressurized air dryer or other drying mechanism to dry the containers 100 faster. In another example, drying the container may be conducted in a separate drying location 902.

[0069] In some aspects, the system 200 may include a sterilization location 904. The sterilization location 904 may be operable to sterilize the container after cells / tissues have been harvested. The sterilization location 904 may have a sterilization system. In some examples, the sterilization system may be an oven to provide dry heat to a container 100 to sterilize it. In another 15 98830719.2Attorney Docket No.121430-791902 example, the sterilization system may be a high-pressure steam system, a hydrogen peroxide fogging system, an ethylene oxide system, or an irradiation system. The sterilization system may include one or more sensors. For example, the oven may include a temperature sensor. The high pressure steam system may include a pressure sensor. The hydrogen peroxide fogging system may include a peroxide concentration sensor. The irradiation system may include a radiation sensor. In some examples, sterilization may be entirely automated. For example, the sterilization system may include a number of automated components such as conveyor belts, rollers, robotic arms, and other automated components known in the art operable to sterilize a container.

[0070] In some aspects, the system 200 may include at least one processor. The at least one processor may be in communication with the one or more sensors. In some examples, the at least one processor may receive data from each sensor and determine a status of each container 100 based on the data provided from each sensor. For example, the status of each container 100 may be one of loading, culturing, ready for harvest, harvesting, cleaning, sterilizing, applying coatings, or annealing. The at least one processor may determine, from data received from a location sensor, that a container 100 is in the cell loading location 910 and label that container 100 as loading. The at least one processor may determine, from data received from a location sensor, that a container 100 is on a shelf of a rack in the climate-controlled room and label that container 100 as culturing. The at least one processor may determine, from data from a location sensor, that a container 100 is in the harvesting location 914 and label that container 100 as harvesting. The at least one processor may determine, from data received from the location sensor, that a container 100 is in the cleaning location 900 and label that container 100 as cleaning. The at least one processor may determine, from data received from a location sensor, that a container 100 is in the sterilization location 904 and label that container 100 as sterilizing. The at least one processor may determine, from data received from a location sensor, that a container 100 is in a coating application location 906 and label that container 100 as coating. The at least one processor may determine, from data received from a location sensor, that a container 100 is in an annealing location 908 and label that container 100 as annealing.

[0071] In some aspects, the at least one processor may be operable to determine a detailed status of each container 100 at each location throughout the system 200. For example, each container 100 may have a volume sensor to determine the amount of loaded cells in each container. The at least one processor may determine a percentage of loading completed or remaining for each container 100 while a container 100 is in the cell loading location 910. The at least one processor may also be configured to determine an amount of time remaining for a 16 98830719.2Attorney Docket No.121430-791902 container 100 being loaded in the cell loading location 910. Once loading has completed for a container 100, the at least one processor may determine that the container 100 is ready to be relocated to the climate-controlled room for culture.

[0072] In some aspects, the at least one processor may be operable to determine a level (e.g., percentage) of tissue growth in a container 100. For example, the one or more sensors may send data to the at least one processor related to parameters of the tissue growth. Once the tissue in a container 100 reach desired parameters, the at least one processor may determine that the container 100 is ready to be harvested. The one or more sensors (e.g., electrical sensor) may detect resistance, capacitance, or impedance by sending an electrical signal through or around the bottom surface 502 of the container 100. The at least one processor may determine the deposited cells and proteins on the bottom surface 502 from the resistance, capacitance, and / or impedance. Once the deposited cells and proteins reach a desired harvesting level, the process is completed. The sensors may be in constant communication (e.g., real time communication) with the at least one processor and the at least one processor may be configured to determine a percentage of tissue growth completed based on the data received from the sensors. In another example, the at least one processor may have a timer for each container 100. The timer may be set to a desired tissue growth time. In some examples, the timer may be set for about 1 day to about 14 days. The timer may begin when the container 100 loaded with cells and a culture media is placed on a shelf 302 of a rack 300 in the climate-controlled room. When the timer is completed (e.g., hits 0 seconds), the at least one processor may determine that the container 100 is ready for harvesting. The at least one processor may also be operable to cause the conditions of the climate-controlled room to change by adjusting humidity, atmosphere, temperature, or other parameters as desired by a user.

[0073] In some aspects, the at least one processor may be operable to determine a level or percentage of the completion of the harvesting. In some examples, at least one processor may determine a level of harvesting completed based on a volume reading from the volume sensor in each container 100. When the volume reading reaches a desired level (e.g., near 0 because all the grown cells / tissue have been harvested), the at least one processor may determine that the container 100 is ready to be cleaned and sterilized.

[0074] In some aspects, the at least one processor may be operable to determine a level of cleaning completed. The at least one processor may be in communication with a pH and conductivity sensors attached to a drain of the cleaning system. The at least one processor may indicate that cleaning is completed when the pH and / or conductivity of the fluid entering the drain 17 98830719.2Attorney Docket No.121430-791902 reaches a desired amount indicative of complete removal of cleaning solution or a proper pH. In another example, the at least one processor may have a timer begins running when a container 100 enters the cleaning location 900. The timer may provide a user with a time remaining in the cleaning process. In some examples, the at least one processor may be operable to turn the cleaning systems described herein on and off depending on a desired cleaning time.

[0075] In some aspects, the at least one processor may also be operable to turn the pressurized air dryer on and off to cause the container 100 to dry. In another example, the at least one processor may be operable to set a drying timer. When the drying timer reaches completion (e.g., 0 seconds remaining), the container 100 may be ready to be moved to the sterilization location 904. In another example, the at least one processor may determine that a container 100 is dry by receiving information from a humidity sensor in communication with the drying system. Once the humidity sensor reaches a desired reading, the drying process may be completed. In some examples, the desired humidity reading is less than 50% humidity, less than 40% humidity, less than 30% humidity, less than 20% humidity, less than 10% humidity, less than 5% humidity, or less than 1% humidity.

[0076] In some aspects, the at least one processor may be operable to determine that the condition of sterilization are met and the process is completed. For example, the at least one processor may determine, from data received from sensors in the sterilization system, a temperature, pressure, concentration, or radiation dose (depending on the type of sterilization system used), of the sterilization. The at least one processor may monitor the sterilization parameters to ensure the parameters remain at a desired level for the entirety of the timer duration. For example, the at least one processor may ensure a temperature of about 160 degrees C to about 200 degrees C, or more for heat sterilization. The at least one processor may ensure that a pressure of about 15 psi to about 40 psi, or more for a high-pressure steam sterilization. The temperature for a high-pressure steam sterilization may be about 121 degrees C to about 150 degrees C. The at least one processor may also have a sterilization timer. The sterilization timer may be set for about 1 minute to about 48 hours, or more. When the timer is completed (e.g., 0 seconds remaining), the at least one processor may determine that the container 100 is ready to be loaded with cells for tissue growth again. The at least one processor may also be operable to control the sterilization by causing the sterilization process to begin once a container 100 enters the sterilization location 904. For example, the at least one processor may turn the sterilization systems described herein on and off based on a desired sterilization time. 18 98830719.2Attorney Docket No.121430-791902

[0077] In some aspects, the at least one processor may be operable to determine a level of coating and / or annealing completed. The at least one processor may use timers and / or volume sensors for the container 100 to determine the level or percentage of coating and / or annealing completed. The at least one processor may also be operable to control the coating and / or annealing processes by applying a desired amount of a coating and / or changing the parameters of the annealing system. The at least one processor may also monitor the temperature and humidity of the container 100 during the annealing process to ensure a desired annealing process is conducted. The at least one processor may also set a timer for the annealing process. The annealing timer may set for about 1 hour to about 48 hours, or more.

[0078] In some aspects, the at least one processor may be operable to actuate (e.g., turn on) the movement mechanism. For example, the at least one processor may have a timer or a schedule set to actuate the movement mechanism at certain times when movement of the container 100 is desired.

[0079] In some aspects, the at least one processor may be operable to actuate the valve on the gas source and allow gas to flow into each container. The at least one processor may receive signals from the sensors indicating that more gas is needed and then cause more gas to be input into the container through the gas inlet port.

[0080] In some aspects, the at least one processor may be operable to control the robotic movement system. For example, the at least one processor may use the determined status of each container and move the container throughout the facility as necessary. When the at least one processor determines that a container 100 is fully loaded with cells, the at least one processor may cause the robotic movement system to move the container 100 from the cell loading location 900 to a shelf 302 on a rack 300 in the climate-controlled room 912. When the at least one processor determines that a container 100 is ready for harvest (e.g., the cells have been fully grown to a desired tissue product), the at least one processor may cause the robotic movement system to move the container 100 to the harvesting location 914. When the at least one processor determines that a container 100 has been fully harvested (e.g., all tissue e.g., meat product has been removed from the container), the at least one processor may cause the robotic movement system to move the container 100 from the harvesting location 914 to the cleaning location 900. When the at least one processor determines that a container 100 has been fully cleaned, the at least one processor may cause the robotic movement system to move the container 100 to the sterilization location 904. When the at least one processor determines that a container 100 has been fully sterilized, the at least one processor may cause the robotic movement system to move 19 98830719.2Attorney Docket No.121430-791902 the container 100 from the sterilization location 904 back to the cell loading location 910. In this manner, the at least one processor may fully automate the culture process. It will be appreciated that the order of movements of a container may be rearranged as desired.

[0081] In some aspects, between the sterilization and loading, the one or more coatings may be applied to the bottom surface (or surfaces) of each container 100. The one or more coatings may be applied and then annealed. For example, if two coatings are to be applied, a first coating may be applied then annealed, then a second coating may be applied and annealed.

[0082] In some aspects, the at least on processor may be operable to determine whether a container 100 has been contaminated. The one or more sensors may include a sensor operable to determine whether a container has been contaminated. In some examples, the contamination sensor may be a pH sensor, a volatile organic gas sensor, a color sensor, or any other sensor operable to determine whether the plurality of cells in a container 100 has been contaminated. The at least one processor may receive data from the contamination sensor and determine whether the plurality of cells in the container 100 are contaminated. If the plurality of cells are contaminated, the at least on processor may remove the contaminated container from its current location (e.g., climate-controlled room 912 or other location) and send the contaminated container to the cleaning location 900 to begin the cleaning and sterilizing process. In this manner, the contaminated container, and the cells / tissues within the container, may be excluded from the harvesting location 914.

[0083] In some aspects, the at least one processor may be in communication with a display. The display may be operable to display a status of each container 100 in the system 200, allowing a user to receive detailed status of each container throughout the tissue growth process. In some examples, the display may also allow a user to input various parameters into the system 200. For example, the user may input the type of cells and / or tissue to be produced in a specific container. The at least one processor may then be programmed to run a certain process depending on the tissue to be produced. In another example, the display may be operable to display an amount of harvested product.

[0084] It will be appreciated that the at least one processor may be operable to control the entire production process such that no physical human interaction is necessary with the components of the system. For example, a user may input desired thresholds, timers, and a desired output and allow the processor to control the entire tissue growth process. 20 98830719.2Attorney Docket No.121430-791902

[0085] In some aspects, the system 200 described herein provides a more efficient and effect solution to large scale tissue production. For example, each container 100 in the system 200 is much smaller than commercially available bioreactors for large scale production. In commercially available bioreactors, if a bioreactor is contaminated, a large amount of product is loss. Conversely, the system 200 described herein solves this issue by providing a plurality of smaller containers 100 for cellular agriculture. Another benefit of the system 200 provided herein is that sterilization may be conducted without the need for high pressure steam methods. Due to the configuration of each container 100, the containers 100 may be sterilized using safer and less expensive techniques. Another benefit of the system 200 provided herein is that the automated systems remove potential contamination for workers who would typically be required to move the containers and load, harvest, sterilize, and clean the containers. Another benefit of the system 200 provided herein is less frequency of movement of containers as compared to continues agitation of other systems, improve the yield of production by reducing the damage to cells via shear stress and increasing the likelihood of extracellular proteins assembly.

[0086] Details of the various applications and stages of cellular agriculture where these systems may be used are provided in the methods section below. II. Methods

[0087] In some aspects, the present disclosure provides a method for large scale tissue culture. As illustrated in FIG.6, the method 600 may include one or more steps. The methods may use the systems described in Section I.

[0088] In some aspects, the method 600 may include cleaning, rinsing, and drying a plurality of containers. The cleaning, rinsing, and drying steps may be conducted using the cleaning system described herein.

[0089] In some aspects, the method 600 may include a sterilization step. The sterilization step may be conducted using the sterilization systems described herein. In some examples, the sterilization step may include one or more of dry heat in an oven, high pressure steam, hydrogen peroxide fogging, ethylene oxide, irradiation, or other sterilization processes known in the art.

[0090] In some aspects, the method 600 may include applying one or more coatings to the bottom surface of the containers. The one or more coatings may be any coating or combination of coatings described herein. After a coating has been placed on the bottom surface of a 21 98830719.2Attorney Docket No.121430-791902 container, the coating may be annealed. A second coating may be applied on top of the first coating and then annealed. More coatings may be applied in the same manner.

[0091] At block 602, the method 600 may include loading a plurality of cells and a culture media into a plurality of containers. The plurality of containers may be the plurality of containers described above. In some examples, the plurality of cells and the culture media may be loaded into the plurality of containers at a depth of about 0.1 cm to about 0.5 cm or 0.1 cm to about 100 cm. The plurality of cells may be mammalian cells, avian cells, fish cells, or other types of cells. In some examples, the plurality of cells may be adherent cells or non-adherent cells. In some examples, the culture media may be fetal bovine serum (FBS), animal plasma, animal platelet rich plasma, egg yolk media, DMEM basal media, or other culture medias known in the art for cells and tissue culture.

[0092] In some aspects, the plurality of cells and the culture media may be mixed with a plurality of microcarriers prior to loading the plurality of cells into the plurality of containers. In an example, a plurality of bare microcarriers may also be loaded into the plurality of containers. The plurality of bare microcarriers may allow immigration of the cells from the mixed cells to the plurality of bare microcarriers. In some examples, the cells and culture media may be mixed with microcarriers at about 2% to about 60%, about 5% to about 30%, or about 20% v / v.

[0093] At block 604, the method 600 may include placing the plurality of containers on one or more shelves of at least one rack of a climate-controlled room. In some examples, the climate- controlled room may control various parameters of the external atmosphere around the plurality of containers. The climate-controlled room is any space that is operable to control climate related parameters. For example, the climate-controlled room may be operable to have a temperature of about 15 degrees C to about 40 degrees C. In some examples, the temperature in the climate- controlled room may depend on the type of cells to be grown. For example, the temperature may be about 37 degrees C for non-primate animal cells, about 38 degrees C for avian cells, about 15-30 degrees C for fish cells, about 20 degrees C for shrimp cells, and about 20-36 degrees C for reptiles. The climate-controlled room may be operable to have an atmosphere of about 85% to about 97% oxygen. In an example, the climate-controlled room may be operable to have an atmosphere of about 3% to about 15% CO2. In some examples, the climate-controlled room may be operable to regulate the humidity in the climate-controlled room. For example, the climate- controlled room may be operable to maintain a humidity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more. In some examples, the humidity is above 90% to avoid water loss in the culture media. 22 98830719.2Attorney Docket No.121430-791902

[0094] In some aspects, the method 600 may include providing motion to each container about every 30 minutes to about every hour via a movement mechanism. The movement mechanism may provide angular change, rotational movement, vibration or combination thereof to each container at a desired time. The movement mechanism may be the movement mechanism described above and may be controlled by the processor as described above. The movement may provide the cells with necessary motion for gas and nutrient exchange. In other examples, the plurality of cells may be cultured in a static or substantially static condition.

[0095] At block 606, the method 600 may include receiving data from one or more sensors connected to or in communication with each container. In some examples, the one or more sensors may be a glucose, lactate, pH, dissolved oxygen, temperature, CO2, or volatile organic gas sensor. In another example, the one or more sensors may further include a location sensor, a movement sensor, a pressure sensor, a peroxide sensor, a volume sensor, or other sensors. In some examples, the one or more sensors may be in communication with at least one processor. The one or more sensors may be operable to send signals (e.g., readouts) from the one or more sensors to the at least one processor. In an example, the one or more sensors may provide real time data to the at least one processor.

[0096] At block 608, the method 600 may include determining, via the processor, that the plurality of cells in a container of the plurality of containers has transformed (i.e., grown) into a plurality of tissues ready for harvesting. The processor may determine that the container is ready for harvesting based on the data received from the one or more sensors.

[0097] At block 610, the method 600 may include moving the container with the plurality of cells and / or tissues that are ready for harvesting to a harvesting location. As described above, the container may be relocated to the harvesting location via an automated robotic movement system or may be manually moved by a human being.

[0098] At block 612, the method 600 may include harvesting the plurality of tissues from the container. Harvesting may include physical force with a harvesting tool, flow washing, scraping, vibrating, sonicating, or other methods of harvesting tissue known in the art. In some examples, harvesting may require less force due to one or more coatings on the bottom surface of the container. The coatings described above may be used to ease the force necessary to harvest the plurality of tissues.

[0099] The method 600 described herein may be repeated as many times as desired. It will be appreciated that the order of the method 600 steps is provided by way of example only. Some 23 98830719.2Attorney Docket No.121430-791902 steps may be completed out of order or reordered as desired. For example, if there is a system failure (e.g., contamination, power loss, or other disruption to the system), the previous step may be repeated, or the system may send the container that experienced the failure back to the cleaning step.

[0100] Further provided herein is a method for processing a container for cellular agriculture. The system, as illustrated in FIG.9, shows the locations the container may move through to be processed. The method may begin at the cleaning location 900 where the container is cleaned as described herein. The container may then move to a drying location 902 where it is dried. In some aspects, the cleaning location 900 and drying location 902 may be the same location. The container may then move to the sterilization location 904 where it is sterilized as described herein. A coating may then be applied to the bottom surface and / or the one or more side surfaces of the container at the coating location 906 as described herein. The coating may then be annealed at the annealing location 908 as described herein. If a second coating is desired, the container may go back to the coating location 906 for the coating to be applied and then to the annealing location 908 again. The coating process may be repeated as many times as desired. The container may then go to the loading location 910 where the plurality of cells and culture media are loaded into the container for tissue growth as described herein. The container may then go to the climate- controlled room 912 where the plurality of cells is grown into a plurality of tissues and proteins as described herein. The plurality of tissues and proteins may then be harvested at the harvesting location 914 as described herein. The method may be repeated as many times as desired. The automated robotic movement system described herein may move the container between the various locations according to a schedule. Each location may have the systems and subsystems described herein which may be fully automated by the at least one processor. Terminology

[0101] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present inventive concept or the appended claims.

[0102] Further, as the present inventive concept is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present inventive concept and not intended to limit the present inventive concept to the specific 24 98830719.2Attorney Docket No.121430-791902 aspects shown and described. Any one of the features of the present inventive concept may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, shape or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present inventive concept may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present inventive concept will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present inventive concept, and be encompassed by the claims.

[0103] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0104] The terms "comprising," "including" and "having" are used interchangeably in this disclosure. The terms "comprising," "including" and "having" mean to include, but not necessarily be limited to the things so described.

[0105] Lastly, the terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this 25 98830719.2Attorney Docket No.121430-791902 definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive. EXAMPLES

[0106] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure. EXAMPLE 1

[0107] In a first example, chicken myofibroblasts were expanded and cultured with the same microcarriers with different mixing conditions for 7 days. The atmosphere content was 5% CO2 and the temperature inside the climate-controlled room was kept at 37 degrees C. After culture, the cells and microcarriers were stained with Calcine-D fluorescent dye that stain live cells and tissue in green. The stained cells / tissue were then examined under a microscope. Table 1 provides the types of mixing conditions and the results of each type of mixing condition. Mixing Condition Results Stirred bioreactor using a paddle Cells had round morphology and colonized in clusters. Some of the clusters were attached to the microcarriers. Overall, the stirred bioreactor using a paddle indicated a stressful culture condition. Stirred bioreactor using a marine impeller Cells had round morphology and colonized in clusters. Some of the clusters were attached to the microcarriers. Overall, the stirred bioreactor using a marine impeller indicated a stressful culture condition. Orbital shaker The microcarrier coverage by cells was better than the stirred bioreactors and the rolling bottle. However, the cells still did not cover all of the microcarriers and round shape cells were observed. Rolling bottle Cells had round morphology and colonized in clusters. Some of the clusters were attached to the microcarriers. Overall, the rolling bottle indicated a stressful culture condition. Shallow static stainless steel tray The cells covered the majority of the microcarriers. Surprisingly, in some areas 26 98830719.2Attorney Docket No.121430-791902 myotubes were formed that resulted in several myoblast fusions. The cell morphology resembled those growing on a 2D surface such as at the bottom of cell culture flasks. Table 1: Results of Different Mixing Conditions

[0108] The results of Table 1 illustrate that overall, the cells grew best in a static condition. The cells grew and formed muscle fibers. Shear stress, especially significant amounts of shear stress, hamper cell growth and muscle formation. The results of the experiment show that static or near static condition for cell growth and muscle tissue formation is preferable. EXAMPLE 2

[0109] Chicken myofibroblasts were cultured in a cell culture dish at a density of 10,000 cells / mL and were left to adhere to a cell culture flask for 12 hours. The cells were then grown for 5 days in egg yolk media composed of Dulbecco’s Modified Eagle Medium (DMEM) and 7% egg yolk in two different conditions.

[0110] The first study group was grown in a static condition and the second study group was grown with an orbital shaker at 30 rpm to measure the impact of shear stress on cell growth and ECM synthesis.

[0111] Cell proliferation was significantly higher in statically grown cells (5,000,000 ^ 200,000) than in the cells grown with the orbital shaker (3,200,000 ^ 350,000) at frequency of 90 rpm. Furthermore, total deposited protein was higher in the statically grown cells (350 ^ 65 mg / dish) than in cells grown with the orbital shaker (144 ^ 83 mg / dish). Normalized protein mass to cell was 70 ng / cell for statically grown cells and 45 ng / cell for cells grown with the orbital shaker.

[0112] The data indicates that even though in both conditions the cells adhere to the bottom of the cell culture dish, static culture conditions are favorable for increasing the mass yield of muscle tissue production by two different mechanisms. First, cell proliferation is improved, and second, deposited cell protein per cell is improved. EXAMPLE 3

[0113] Chicken myofibroblasts were cultured in a cell culture dish at a density of 10,000 cells / mL and allowed to adhere to a cell culture flask for 12 hours. The cells were then grown for 10 days in egg yolk media composed of Dulbecco’s Modified Eagle Medium (DMEM) and 7% egg yolk in two different conditions. 27 98830719.2Attorney Docket No.121430-791902

[0114] The first study group was grown on a flat culture dish and the second study group was grown on a grooved substrate with cross-sectional groove profile of triangles at 1.4 millimeter wavelength and 1 millimeter wavelength to determine the impact of groove substrate on tissue growth.

[0115] After the culture period, the formed tissue was physically scraped from both substrates. The mass of the collected tissues was determined and normalized to the cultured surface area. The results showed that the harvested tissue grown on the grooved substrate was 2.5-3.5 g per square decimeter, while the flat substrate resulted in 0.05-0.15 g per square decimeter. Furthermore, upon visual examination, tissue detachment and wrapping were observed in several areas of the tissue culture surface area for the tissue grown on the flat substrate, while no detachment was observed for the tissue grown on the grooved substrate. 28 98830719.2

Claims

Attorney Docket No.121430-791902 CLAIMS What is claimed is:

1. A method for large scale tissue culture, the method comprising: loading a plurality of cells and a culture media into a plurality of containers at a depth of about 0.1 cm to about 100 cm, each container comprising: a bottom surface; one or more side surfaces extending from and surrounding the bottom surface; a top opening; a lockable lid; and one or more sensors; placing the plurality of containers containing the plurality of cells and the culture media on one or more shelves of at least one rack or on a moving conveyer belt in a climate-controlled room; receiving, via a processor, data from the one or more sensors; determining, via the processor, that the plurality of cells in a container has grown to a plurality of tissues for harvest; moving the container with the plurality of cells and / or tissues ready for harvesting to a harvesting location; and harvesting the plurality of cells and / or tissues from the container with the plurality of cells and / or tissues ready for harvesting.

2. The method of claim 1, wherein the plurality of tissues further comprises extracellular proteins.

3. The method of claim 1, wherein each container has an outer bottom surface operable to stack to the lockable lid of another container and the container shape is square, rectangular, circular, any other shape, or any combinations thereof operable to promote cell growth and tissue formation.

4. The method of claim 1, wherein the plurality of cells is cultured in a static condition.

5. The method of claim 1, the method further comprising providing motion to each container about every 30 minutes to about every hour via a movement mechanism, wherein the 29 98830719.2Attorney Docket No.121430-791902 movement mechanism provides angular change, rotational movement, vibration or combinations thereof to each container.

6. The method of claim 1, the method further comprising: mixing the plurality of cells with a plurality of microcarriers prior to loading the plurality of cells into the plurality of containers; and loading a plurality of bare microcarriers into the plurality of containers prior to moving each container to the climate-controlled room; wherein the plurality of bare microcarriers allow immigration of the cells from the mixed cells and microcarriers to the plurality of bare microcarriers; wherein the plurality of cells mixed with microcarriers are about 2% to about 60% v / v.

7. The method of claim 1, wherein the bottom surface is patterned with grooves to increase surface area and / or induce cell alignment and / or accelerate and promote tissue growth.

8. The method of claim 7, wherein grooves have a cross-sectional profile selected from triangle, square, sinusoid, semi-circle, similar geometries, or combinations thereof to accelerate and improve the cell and tissue growth.

9. The method of claim 8, wherein the amplitude of cross-section groove profile is about 0.5 to about 5 millimeters profile wavelength is about 0.5 to about 5 millimeters.

10. The method of claim 1, wherein the bottom surface is patterned with void and / or wells and / or meshwork with different shapes or geometries to increase surface area and / or induce cell alignment and / or accelerate and improve tissue growth.

11. The method of claim 1, wherein the bottom surface is patterned with vertical posts to support three-dimensional tissue formation.

12. The method of claim 1, wherein the bottom surface has a rough surface to increase surface area and harvestable yield of cells and tissues.

13. The method of claim 1, the method further comprising applying a first coating to the bottom surface of each container prior to loading the plurality of cells into each container, wherein the first coating is cell and protein non-adhesive. 30 98830719.2Attorney Docket No.121430-791902 14. The method of claim 13, the method further comprising applying a second coating on top of the first coating, wherein the second coating is configured to preserve ECM proteins.

15. The method of claim 13, the method further comprising applying a second coating on top of the first coating, wherein the second coating is cell adhesive proteins, wherein the cell adhesive proteins are one or more of gelatin, collagens, fibronectin, elastin, vitronectin, or other proteins from natural or recombinant sources.

16. The method of claim 1, the method further comprising applying a first coating to the bottom surface of each container prior to loading the cells into each container, wherein the first coating is cell adhesive proteins, wherein the cell adhesive proteins are one or more of gelatin, collagens, fibronectin, elastin, vitronectin, or other proteins from natural or recombinant sources.

17. The method of claim 16, wherein adhesive proteins are obtained from collagenous membranes of egg-shells by hydrolyzing in acidic or basic conditions, mechanical or biochemical digestion, or combinations thereof.

18. The method of claim 1, the method further comprising applying a first coating to the bottom surface of each container prior to loading the cells into each container, wherein the first coating is cell adhesive proteins extracted from a prior cell culture of the same species.

19. The method of claim 13, the method further comprising applying a second coating on top of the first coating, wherein the second coating is cell adhesive proteins extracted from a prior cell culture of the same species.

20. The method of any one of claims 13-19, the method further comprising functionalizing the first and / or second coating to guide the cells and provide preferential tissue formation, wherein the first and / or second coating is functionalized using a laser, microcontact printing, molding, stamping, chemical reactions, or combinations thereof.

21. The method of claim 1, the method further comprising: moving each container to a sterilization location prior to loading each container with the plurality of cells and / or after harvesting the plurality of cells and / or tissues from each container; and 31 98830719.2Attorney Docket No.121430-791902 sterilizing each container, wherein sterilizing each container comprises one or more of dry heat in an oven, high pressure steam, hydrogen peroxide fogging, ethylene oxide, irradiation, or combinations thereof.

22. The method of claim 1, wherein the climate-controlled room has a temperature of about 15 degrees C to about 40 degrees C, a humidity level of at least 80%, and an atmosphere of about 85% to about 97% oxygen and about 3% to about 15% CO2.

23. The method of claim 1, wherein the harvested cells and / or tissues are muscle tissue or meat products.

24. The method of claim 21, wherein moving each tray between the sterilization location, climate-controlled room, and / or harvesting location is conducted by an automated robotic system, conveyer, or manually by a human.

25. The method of claim 20, wherein the processor controls the automated robotic system and causes the automated robotic system to move each container between the sterilization location, climate-controlled room, and / or harvesting location based on data from the one or more sensors.

26. A system for large scale cellular agriculture, the system comprising: a plurality of containers comprising: a bottom surface; one or more side surfaces extending from and surrounding the bottom surface; a top opening; one or more sensors; and a lockable lid; a climate-controlled room; at least one rack having a plurality of shelves in the climate-controlled room, each shelf operable to hold at least one container of the plurality of containers; a movement mechanism operable to provide a force to each container; and at least one processor in communication with the one or more sensors, the processor configured to: receive data from the one or more sensors; determine a status of each container; and 32 98830719.2Attorney Docket No.121430-791902 cause motion of each container about every 30 minutes to about every hour via the movement mechanism; wherein each container has a volume of about 1 L to about 1000 L; wherein each container is configured to contain a plurality of cells in a culture media.

27. The system of claim 26, wherein the movement mechanism provides angular change, rotational movement, vibration, or combinations thereof to each container.

28. The system of claim 26, wherein each container is made of or coated with polymers, hydrogels, glass, ceramics, metals, or combinations thereof.

29. The system of claim 26, wherein each container is made of stainless steel.

30. The system of claim 26, wherein the bottom surface has a first coating that is cell and protein non-adherent.

31. The system of claim 30, wherein the bottom surface has a second coating on top of the first coating configured to preserve ECM proteins.

32. The system of claim 26, wherein the lockable lid has a filter screen made of natural polymers, synthetic polymers, ceramics, metals or combinations thereof and having a plurality of pores with a diameter of less than 1 µm or a gas permeable silicon membrane.

33. The system of claim 26, wherein the lockable lid has an opening to an external atmosphere for gas exchange and the opening is configured to prevent entrance of free-floating particles into the container.

34. The system of claim 26, wherein the climate-controlled room has a temperature of about 15 degrees C to about 40 degrees C, a humidity level of at least 80%, and an atmosphere of about 85% to about 97% oxygen and about 3% to about 15% CO2.

35. The system of claim 26, wherein each container is operable to contain a plurality of microcarriers carrying the plurality of cells and a plurality of bare microcarriers, wherein cells immigrate between the microcarriers carrying cells and the bare microcarriers.

36. The system of claim 26, wherein the one or more sensors are glucose, lactate, pH, dissolved oxygen, temperature, CO2, and / or volatile organic gas sensors. 33 98830719.2Attorney Docket No.121430-791902 37. The system of claim 26, wherein the bottom surface of each container is patterned with grooves to increase surface area and induce cell alignment and improve tissue growth.

38. The system of claim 26, the system further comprising an automated robotic movement system operable to move each container between a cell loading location, the climate- controlled room, a harvesting location, a sterilization location, and a cleaning location.

39. The system of claim 38, wherein the at least one processor is configured to control movement of each container by the automated robotic movement system based on the data received from the one or more sensors and the status of each container.

40. The system of claim 38, wherein the automated robotic movement system reduces risk of contamination by at least 50% or more.

41. The system of claim 40, wherein the one or more sensors are operable to detect a contamination of each container before moving each container to the harvesting location and wherein when a container is contaminated the container is excluded from the harvesting location.

42. The system of claim 26, wherein the status of each container is one of loading, culturing, ready for harvest, harvesting, sterilizing, cleaning, coating, or annealing.

43. The system of claim 26, wherein a container is processed by a method comprising: cleaning and rinsing the container; drying the container; sterilizing the container; applying a first coating to the container; annealing the first coating onto the container; applying a second coating to the container; annealing the second coating onto the container; loading the container with a plurality of cells and a culture media; culturing the plurality of cells in the container in the climate-controlled room; harvesting a plurality of tissues and proteins grown from the plurality of cells; and repeating the method a desired number of times. 34 98830719.2

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