APPARATUS AND PROCESS FOR CULTURING TISSUE

An apparatus for culturing muscle tissue comprises a chamber extending from a first end to a second end; a first anchor located within the chamber at the first end; a second anchor located within the chamber at the second end; an inlet; and an outlet. The chamber can have an expanding or cylindrical cross-section from each of the first end and the second end to a point between the first end and the second end. The first and second anchors are three-dimensional.

FIELD OF THE INVENTION

The present invention relates to producing tissues from cells, and more specifically an apparatus and methods for production of muscle tissue.

BACKGROUND

The term cultured meat, also called in vitro meat, lab-grown meat, cell-based meat, cultivated meat and synthetic meat is used to refer to muscle tissue grown from a cell or tissue culture as opposed to slaughtering animals to obtain the muscle tissue. This is desired to reduce the environmental impact of the agricultural system, as well as to address animal welfare issues and keep up with the growing demand for meat in the world.

The first attempts at cultured meat production have thus far used many of the same tissue engineering techniques used in regenerative medicine, producing small amounts of tissue in small bioreactor chambers with labor intensive processes.

SUMMARY

According to a first aspect of the invention, an apparatus for culturing muscle tissue comprises a chamber extending from a first end to a second end,; a first three-dimensional anchor located within the chamber at the first end; a second three-dimensional anchor located within the chamber at the second end; an inlet; and an outlet. Such an apparatus can culture large pieces of muscle tissue between the three-dimensional anchors as the three-dimensional anchors allow for a plurality of attachment points for secure connections, allowing for more distance between then anchors than past systems and therefore larger muscle tissues.

In some embodiments, the chamber can have an expanding cross-section from each of the first end and the second end to a point between the first end and the second end. Such an apparatus with a chamber which has an expanding cross-section from each end to a middle point (for example, barrel or oval shaped) results in the ability to produce muscle tissue in large, desirable shapes when working with tissue which compacts. Having a chamber with an expanding cross-section to a middle point results in the muscle tissue grown in the chamber having a consistent, or even a convex shape between anchors after compaction, producing more muscle tissue and more closely mimicking muscle tissue produced in animals.

According to an embodiment, each of the first anchor and the second anchor have a biomimetic shape, for example, modelled after a tendon. Optionally, the three-dimensional anchor comprises an outer perimeter and a plurality of attachment paths extending from the outer perimeter. Further optionally, the outer perimeter is cylindrical, and/or the plurality of attachment paths do not align in the vertical direction. In further embodiments, the attachment paths may align in the vertical direction.

According to an embodiment, each three-dimensional anchor is an auxetic design. Auxetic design, as used herein, means that the anchor is formed of three dimensional building blocks which, when a force is applied in one direction results in an expansion in a perpendicular direction to the direction of the applied force. Optionally, the three-dimensional anchor is formed of a plurality of auxetic building blocks connected together. The building blocks could be, for example, butterfly shaped hexagon prisms connected at a center point which can deform into square or rectangular shapes. Further optionally, the anchor is a square or rectangular configuration with a plurality of building blocks connected together in at least two horizontal planes, each of the horizontal planes being connected to each other vertically. The use of relatively small auxetic building blocks providing small pores has been shown to result in a positive influence in the attachment of the tissue to the anchor, and the auxetic design allows for the use of these small building blocks in a repeated manner to build up the anchor to any size required which maintaining many attachment points and strength. The auxetic design provides for additional strength, even when subject to relatively high mechanical stimulation loads, with forces in one direction resulting in elongation in another.

In some embodiments, one or more of the anchors can be formed of a conductive material, for example metals (including alloys) such as titanium, ferritic steel, iron and carbon. Such a three-dimensional anchor design with a plurality of attachment paths create a plurality of pores in the anchors where the muscle tissue is able to wrap around and attach to many different points and sides of the attachment paths or auxetic building blocks and the perimeter of the anchors. This results in stronger connections between then anchors and muscle tissue, allowing for the growth of larger muscle tissue and for more mechanical stimulation, resulting in more efficient growth and muscle tissue quality in terms of texture, appearance and taste. The use of conductive material to form anchors allows for the direct connection and/or integration of one or more electrodes into the apparatus to provide electrical stimulation to chamber, to promote cell differentiation and tissue compaction as well as muscle tissue growth.

According to an embodiment, the chamber cross-section is circular, oval, rectangular or triangular. Other chambers could contemplate other designs as well, such as octagon or another polygonal cross-sectional shape, which may be useful for manufacture. A circular or oval cross-section can produce muscle tissue in a consistent or even convex shape, thereby producing more and larger pieces of tissue than past systems, as well as tissue which more closely resembles muscle tissue grown in animals.

According to an embodiment, the chamber can be up to 1-2 L in volume, preferably, 250 mL-1.5 L, further preferably 500 mL to 1 L. The use of a chamber with expanding cross-section and three dimensional anchors allows for much larger chambers than seen in the prior art, thereby promoting more efficient muscle tissue production.

According to an embodiment, when the chamber expands in cross-section from each of the ends to a middle point, the middle point is half way between the first end and the second end. Such a design can form a chamber that is substantially symmetric along a horizontal plane at its middle point.

According to an embodiment, the chamber is symmetrical along a central vertical axis between the first end and the second end. This can be useful for efficiency of design and manufacture of the chamber.

According to an embodiment, the first anchor and/or the second anchor are moveable. Optionally, the first anchor and/or the second anchor are movable in a vertical direction and/or rotatable. Such movement(s) can be associated with one or both anchors. For example, one anchor could be rotatable, and one anchor could be moveable in the vertical direction, thereby respectively providing various types of mechanical stimulation to the muscle tissue. Each movement could be powered by separate motors located at or near anchors or connections. Using separate motors could allow for smaller motors, thereby helping to ensure apparatus stays compact overall. Separate motors could also ensure that not all mechanical stimulation ceases should one motor fail. Motors can be electrical and/or battery powered. Making anchors moveable allows for a simple way of providing mechanical stimulation to muscle tissue, helping to promote efficient growth and development of muscle tissue. Using rotational and vertical stimulation allows for putting tension on the muscle tissue in all directions (x, y and z) through various movements, while the anchors ensure that the muscle tissue stays connected throughout the stimulation. In some embodiments, one motor could be used to power both anchors and/or only one anchor could provide both rotational and vertical movements.

According to an embodiment, the first anchor and/or the second anchor are removable from the chamber. These can be through quick release couplings, allowing for simple removal of muscle tissue with anchors upon maturation of the tissue. Such couplings could be, for example, magnetic couplings where a base of the anchor and/or a cap holding the anchor connects to an electromagnet which can be switched on to secure the anchor in place and turned off to remove the anchor/cap with the anchor. Such removable anchors can also help for the cleaning of anchors for reuse (e.g., inserting into an autoclave), or to allow efficient disposal and replacement when anchors are not reusable.

According to an embodiment, the apparatus comprises a heating element. Optionally, the heating element is configured to control a temperature of fluid within the chamber. The heating element can be, for example, heating wires embedded directly in the walls of the chamber to heat up the medium in the chamber through the surface. Optionally, a thermoprobe or other means of sensing the temperature could be used to ensure the temperature inside the chamber and directly surrounding the tissue is precisely controlled. Typically, the temperature for developing muscle tissue would be around 37 degrees C., though could be 32-42 degrees C., or in some cases even outside this range. A further option, in addition or alternative to heating wires could include a jacket around the chamber. Such a jacket could simply be an insulating jacket to help control heat loss, or could surround chamber and have a controlled temperature fluid flow between the jacket and chamber to precisely control the temperature inside of the chamber, thereby controlling conditions for optimal tissue growth and maturation, as well as for the precise temperatures needed for the cooling down and harvesting process.

According to an embodiment, the apparatus further comprises one or more electrodes for providing electrical stimulation. These can be connected, for example, to the first anchor and/or the second anchor for providing the stimulation, though could be connected elsewhere. By directly connecting and/or integrating one or more electrodes into the apparatus, the apparatus is able to provide electrical stimulation to chamber without extra outside parts, thereby promoting cell differentiation and compaction as well as muscle tissue growth.

According to an embodiment, the apparatus could have a manual or automatic control system (or partially automatic and partially manual) to control various aspects of apparatus and the process, such as temperature, pH, flow through the chamber, dissolved oxygen, glucose, etc. Such a control system could connect to sensors, and have various alerts or alarms to warn of conditions which are not ideal or malfunctioning hardware. Such sensors and/or alarms could also indicate the various stages of maturation and alert to maturity markers. Such a system could include various hardware and/or software components, such as one or processors (which could be a special purpose processor for apparatus), memory, a user interface, etc. which could be located at apparatus10, or remotely (e.g., through a wireless or wired connection). In some instances, one control system could control a number of apparatuses, each apparatus growing tissue.

According to an embodiment, the inlet is located in a shower cap comprising a plurality of outlets configured to flow media into the chamber from an upper end of the chamber at a plurality of points surrounding a central axis of the chamber. Optionally, the shower cap comprises the inlet for receiving the media, a channel for distributing the media around a central axis, and a plurality of outlets connected to the channel for flowing the media out of the channel and into the chamber. Such a design results in outlets being generally located around the circumference or perimeter of the top anchor (below the shower cap) and the eventual tissue. The media flows out of outlets into chamber all around the top anchor and tissue, thereby providing for better flow and distribution of media into and around the tissue and chamber. This leads to better mixing in the chamber as well as better perfusion.

According to a further aspect of the invention, an anchor for a tissue reactor chamber comprises a connector; and a three-dimensional projection extending from the connector. Optionally, this connector can be a connector rod which connects to or through the three-dimensional projection. Further optionally, the three-dimensional projection comprises an outer perimeter extending longitudinally, and a plurality of attachment paths extending from the outer perimeter. Further optionally, the plurality of attachment paths do not align in the longitudinal direction. In other embodiments, the plurality of attachment paths do align in the longitudinal direction. Optionally, the anchor is formed of conductive material and/or food safe biomaterial.

Such a three-dimensional anchor provides a secure point to which muscle tissue can connect when developing in a tissue reactor chamber. The use of a three-dimensional projection with a plurality of attachment paths ensures a large network of points in many directions where the muscle tissue can attach to the anchor, thereby ensuring the connection can withstand stress and tension in all directions (e.g., x, y and z). This allows for mechanical stimulation to help promote efficient tissue growth and maturation as well as quality texture in the muscle tissue produced. Using conductive material allows for electrical stimulation directly through the anchor, and a food-safe biomaterial can help to promote the tissue attachment and growth while ensuring that the material does not otherwise affect the tissue in terms of flavour, look, taste, etc.

According to a further aspect of the invention, a method for growing tissue in a chamber comprises filling a chamber with a suitable scaffolding material comprising cells, the chamber with a first three-dimensional anchor located at a first end and a second three-dimensional anchor located at a second end; and incubating the scaffolding material comprising the cells under conditions suitable for maturation of the cells, whereby the mature cells form tissue connecting to and between the first and second anchors. Such a suitable scaffolding material can optionally be a hydrogel. Optionally, the chamber can be barrel shaped or cylindrical. The filling can be done through an inlet, through the top or any other suitable method.

Such a method is able to produce large pieces of muscle tissue in the chamber between the anchors. In some embodiments, the chamber is substantially barrel shaped. When the chamber barrel shaped, it is especially useful for the production of compacting tissue. The barrel shape is used in this context to refer to a chamber that is expanding in cross-section to have the largest cross-section at a point between the ends. It could be curved, or could come to an apex at the largest cross-section. It was surprisingly discovered that muscle tissue produced with such a process would be either consistent in cross-section between the anchors or even convex, producing more tissue and more closely resembling muscle tissue grown in animals when growing tissue which compacts.

According to an embodiment, the method further comprises providing mechanical stimulation through rotational and/or vertical movement of at least one of the first anchor and the second anchor. Optionally, the method further comprises providing electrical stimulation through one or more electrodes connected to at least one of the first anchor and the second anchor. Such mechanical and/or electrical stimulation can help to more efficiently differentiate as well as elongate and align cells. It can also help to compact the tissue, as well as produce and mature muscle tissue after compaction. In some instances the mechanical and/or electrical stimulation could even be used as a maturity indication (e.g., the tissue has a certain resistance when mature). Due to the use of three-dimensional anchors, greater amounts of mechanical stimulation can be provided without the risk of tearing tissue from anchors, for example, inducing a strain of about 30, preferably 20-100.

According to an embodiment, the step of incubating the scaffolding material comprising the cells under conditions suitable for maturation of the cells, whereby the mature cells form tissue connecting to and between the first and second anchors comprises controlling at least one of the following in the chamber: temperature, pH, dissolved oxygen and glucose; and/or flowing liquid through the chamber. Optionally, liquid can be flowed through the tissue. Such a method uses perfusion and/or circulation around the tissue to promote efficient muscle tissue growth and maturation. Flowing liquid around and/or through the tissue can be done by ensuring there is a constant flow of liquid into the inlet of the chamber and out of the outlet. This can be very slow, for example, 1-2 ml/minute, though could vary, for example, up to 20 mL/minute, with liquid flow rate inside the tissue being lower than around the tissue. Such flow runs growth media around the tissue and into the tissue, promoting healthy growth of muscle tissue in the chamber. The mechanical stimulation can also help to promote the waste media exchange, allowing for the infusion of fresh medium into the tissue. The temperature and/or pH can be controlled with various sensors and/or other components or devices to ensure the correct conditions for promotion of muscle tissue growth and development. Temperature control can be through heating wires directly in chamber wall, an outer jacket (which could include a fluid flow) or other methods.

According to an embodiment, the cells are pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells. Optionally, the mature cell is a myocyte. In certain embodiments, the mature cells are a mixture of myocytes and adipocytes.

According to an embodiment, the tissue is muscle tissue. Such a method is ideal for growing muscle tissue, particularly that from animals for consumption. In certain embodiments, the muscle tissue further comprises fat tissue.

According to a further aspect, the invention provides for a method of manufacturing engineered tissue products by using the apparatus as described herein. In some embodiments, the engineered tissue products are muscle tissue are intended for research or therapeutic purposes. In certain preferred embodiments, the engineered tissue is muscle tissue intended for dietary consumption by human beings, non-human animals or both. In some embodiments, the engineered muscle tissue products are human food products. In some embodiment, the engineered muscle tissue products are designed to resemble traditional meat products and the cell types are chosen to approximate those found in traditional meat products. Human beings traditionally eat several type of animal muscle tissue. Therefore, in some embodiments, the myocytes are skeletal myocytes or smooth myocytes.

Fat plays a part in the development of the traditional meat flavor during cooking and plays a role in giving meat its characteristic juiciness be enhancing the water-holding capacity of the meat. Accordingly, in one embodiment, the engineered muscle tissue further comprises fat. Therefore, in some embodiments, the muscle tissue further comprises adipose cells.

According to a further aspect of the invention, a chamber for culturing muscle tissue comprises a first end; a second end; and a body with an expanding cross-section from each of the first end and the second end to have a largest cross-section at a point between the first end and the second end. Optionally, the chamber further comprises an inlet and/or an outlet. Further optionally, the body is circular or oval in cross-section. In some embodiments, the chamber has a smooth curvature from each of the ends to the point with the largest cross-section.

Such a chamber can promote the culturing of large amounts of compacting muscle tissue in a desired shape. By having an expanding cross-section from each of the ends, the muscle tissue can compact in a cylindrical or even convex shape, more closely resembling muscle tissue from animals and producing larger pieces of muscle tissue upon compaction.

DESCRIPTION

Base18acts to support and stabilize chamber12, anchors14,16and other supporting parts not shown (motor(s), electrode(s), pump(s), etc.). In the embodiment shown, the chamber is supported from base18, extending longitudinally from holder36at first end22to cap28on second end24of chamber12. Other embodiments, for example, chambers which are not supported from the ground (e.g., suspended) could have different support configurations, changing and/or eliminating one or more of base18, and cap28. Cap28and connections with chamber12can be better seen inFIG.2A, and the exploded view inFIG.1B.

Each of anchors14,16secure to electromagnets29for positioning within chamber12. Anchor14connector26is positioned within cavity33, which can be a blind hole (or a through cavity in some cases) in proximity to electromagnet29at holder36. Metal sink38acts as a heat sink, and fan34helps to blow the heat away so it does not affect the chamber12temperature or the muscle tissue culturing. Holder36helps to position anchors14,16at a sufficient distance from electromagnet29so that the magnetic field generated does not affect the muscle tissue grown. Cap28can connect to chamber12at second end24, with anchor16connecting to electromagnet29through cap28. Cap28can be configured to allow anchor16to move freely. Cap28can also have a fan inside, which is not shown in the Figures. In some embodiments, cap28could be formed integral with chamber12. Cap28could also be used to house other parts of apparatus10, such as motors for mechanical stimulation, electrodes, sensors, alarms, etc. In some embodiments, cap28could be hollow to allow for easy removal of muscle tissue with anchors through one end of chamber12and cap28. Alternatively, the cap28could be removable with the anchor16and tissue at the time of harvest.

Chamber12, which is depicted in more detail inFIGS.2A-2B, extends from a first end22to a second end24along vertical axis A and connects to holder36of base18at first end22and second end24to cap28. Chamber12has an outer wall that expands in cross-section from each of the first end22and the second end24to have a largest cross-section at a point between the first end22and the second end24. This point should be about half way between the first anchor14and the second anchor16, which will often correspond to the half way point between the first end22and second end24, though this could differ depending on anchor14,16placement within chamber12. The shape is generally curved, for example, barrel or oval shape between the connecting ends22,24, though does not have to be curved (e.g., could be conical or slanting from both ends). Chamber is typically formed of a transparent material, such as glass or plastic to enable viewing through the chamber. In some embodiments, chamber could have only limited or no viewing windows (e.g., made of stainless steel), which may help with maintaining temperature, consequently leading to energy savings. Chamber can also include various heating elements and/or temperature control devices, such as heating wires and a thermoprobe, which will be described in more detail.

Chamber12includes an inlet30and outlet32. Inlet30and outlet32allow for controlling the flow of fluid into and out of chamber12. Inlet30and outlet32placement are shown for example purposes only and could be located at different points in the chamber. Some embodiments could have more than one inlet and/or outlet, for example, a large inlet and outlet used only for initial filling of chamber12and draining at harvest with a smaller inlet and outlet for flow during tissue growth processes. An alternative inlet is depicted inFIGS.5A-5B.

Anchors14,16are biomimetic anchors, which are each located within chamber12at opposite ends. The specific design of anchors14,16will be discussed in more detail in relation toFIGS.3A-3B. Anchors14,16are formed of conductive material, for example metals such as Titanium or stainless steel and/or carbon based materials such as graphite and graphene. At least one of anchors14,16are connected to an electrode or other device to provide electrical stimulation to the chamber through the anchor(s). The electrode or other device could be connected to the anchor(s) through the connecting rod(s)26. In some embodiments, electrodes are integrated into the connector rods26or anchor14,16to provide electrical stimulation without the need for separate electrodes and attachments. Anchor14can rotate, with power provided by a connected motor (typically located at an end of connector rod26in cap28). Anchor16can move up and down along the vertical axis A, with power provided by a separate motor (also not shown, but can be located within cap28). In some embodiments, anchor14could move vertically and/or anchor16could rotate. Additionally, some embodiments could power both anchor14,16movements with one motor, though having separate motors can reduce the risk of unintentionally ending all stimulation or movement due to motor failure and allow for smaller motors within apparatus10. In further embodiments, only one of anchors14,16could perform both the longitudinal and rotational movement. Motors can be battery powered and/or electric.

Apparatus10functions to grow muscle tissue in chamber12between anchors14,16. Anchors14,16are placed within chamber, with electromagnets29securing anchors14,16in place. Then chamber12is filled with a hydrogel (or other suitable scaffolding material) through the top and/or inlet. The chamber interior is typically filled fully with the hydrogel with suspended cells.

The cells used could be a number of different cells. In one embodiment, the cells are preferably self-renewing cells such as pluripotent stem cells, or any type of muscle cell. Pluripotent stem cell include embryonic stem cells or induced pluripotent stem cells (iPSC) that maintain the capacity to self-renew in the undifferentiated state, or alternately differentiate to any tissue lineage. In certain embodiments, the cells originate from an animal species intended for dietary consumption, including livestock, poultry and game. In a preferred embodiment, the cells are from bovine or porcine origin. In certain aspects, the cells originate from species intended for research or therapeutic purposes such as humans, primates and rodents including rats and mice.

Chamber conditions are precisely controlled to promote the differentiation and subsequent compaction and maturation of the cells. Typically, the chamber would be kept at about 37 degree C., and pH, dissolved oxygen and/or glucose would be controlled. Culture conditions suitable for the differentiation of pluripotent cells and subsequent maturation can be maintained within the chamber. Cells would then begin differentiating, resulting in tissue compaction. Compaction would typically take about 1-14 days, though this depends on the type of cells, chamber conditions, hydrogel, etc. The cells compact to around half their original volume and begin to attach themselves to anchors14,16at many points around the three-dimensional pathways through anchors14,16. During compaction, cells interact with the hydrogels, pulling fibers and expelling water to compact together and form muscle tissue attaching to and between anchors14,16. Compact cells then mature into muscle tissue, typically in about 12-18 days.

Electrical stimulation through the connection of at least one electrode to at least one of the anchors14,16can begin as soon as the hydrogel fills the chamber12, even before compaction as it can help with cell differentiation, stimulating cells to align, fuse and connect. Such electrical stimulation can help to promote compaction with the cells and promote muscle tissue growth and maturation after compaction.

Mechanical stimulation through movement of one or more anchors14,16can begin after compaction, when muscle tissue has attached to each of anchors14,16. Sometimes a waiting period after compaction is desirable, for example, two days, to ensure there is a strong connection to anchors14,16and that the tissue has developed sufficient integrity and strength to endure the movements before starting mechanical stimulation. The mechanical stimulation can include rotational movement through anchor14and/or vertical movement through anchor16and/or anchor14. Movements must be tailored relative to the size, and typically should not result in strain more than 0.3, preferably no more than 0.2. The three-dimensional anchors14,16with a plurality of attachment paths allow for larger mechanical stimulation due to the stronger attachment of the tissue in x, y and z directions to the anchors14,16.

Chamber12also promotes tissue growth by perfusion (liquid inside the tissue) and perifusion (liquid flowing around the tissue). Specifically, fresh liquid (e.g., water) is flowed through chamber12inlet30and out of outlet32at a constant rate. In some embodiments, the flow could be through separate inlets and outlets designed specifically for perifusion (e.g., sides or ends of the chamber at places where the tissue will reside to ensure flow all around the tissue). This flow around the tissue is typically a rate of around 10 mL-100 mL per minute, with perfusion flow at a very low rate, for example, 0.5-10 mL per minute. Such flow runs culture media around the tissue and into the tissue, promoting healthy growth of muscle tissue in chamber12. The mechanical stimulation, for example, rotation, can also help to promote perfusion, allowing for the infusion of new medium into the tissue, and expelling exhausted media out of the hydrogel. The temperature, pH, dissolved oxygen and glucose are typically controlled with various sensors and/or other components to ensure the correct conditions for promotion of muscle tissue growth and development. The temperature control can be through heating wires directly in chamber12wall, an outer jacket (which could include a fluid flow) or other methods.

After about 12-18 days, muscle tissue develops into a mature shape connected to and extending between anchors14,16. Once the muscle tissue has reached a point of maturity, the tissue can be removed from the chamber12in a harvesting operation. Maturity can be determined in a number of ways, for example, testing of mechanical strength, resistivity, color, density, biomarkers such as myoblast determination protein, etc. Devices and/or sensors which form a part of apparatus10(e.g., mechanical or electrical stimulation components) could be used for testing and recognizing maturity, or outside sensors can be used. Maturity marker testing can be done manually, or automatically, for example, through a control system.

Chamber12could then be flushed with water, or simply drained through outlet32and can be sterilized, for example, in an autoclave. Electromagnets29can be deactivated to release anchor14from base18. Other embodiments could be released and/or decoupled through a plug, button or other quick release mechanism either in place of or in addition to electromagnet19. Anchor16(and all or part of cap28) also has a quick release coupling, deactivating electromagnet29, which allows anchor16to be released from chamber12. Anchors14,16with muscle tissue are extracted from chamber12vertically upwards along vertical axis A through second end24of chamber12. This can be manually or through a machine, arm, or other device. The grown tissue connected at one end to anchor16and at the other end to anchor14moves with anchor16(also bringing anchor14) out of the top of chamber12. Once removed from chamber12, tissue can be removed from anchors14,16, e.g., by cutting.

Apparatus10, through the use of a specific chamber12shape and three-dimensional, biomimetic anchors14,16is able to efficiently produce large, three-dimensional muscle tissue. Past systems for growing muscle tissue in a chamber typically either grew very small amounts of tissue (e.g., 500 microliters to 2 milliliters) or two-dimensional tissue. In some systems where three dimensional tissue was grown, it would typically connect to anchor points and have a very thin connection in the middle of anchor points with a concave or hourglass shape. The shape of chamber12allows for the growth of muscle tissue with a substantially uniform cross-section, or even a convex shape between anchors14,16, resulting in more tissue produced and a more desirable muscle tissue shape which more closely resembles meat from animal. This shape is typically more desirable for use (being larger) and for consumer preferences.

The use of three-dimensional biomimetric anchors14,16promotes stronger connections of muscle tissue to anchors, thereby allowing for efficient production of larger quantities of muscle tissue, as well as increased mechanical stimulation resulting in muscle tissue with increased quality in terms of texture, taste and visual appearance. The three-dimensional biomimetric anchors14,16also allow for a large amount of connection points for muscle tissue with anchors14,16, resulting in decreased risk of the muscle pulling away from anchors14,16, even during relatively strong mechanical stimulation. The ability to easily remove anchors14,16with quick coupling mechanisms also ensures a simple and efficient harvest operation. Additionally, the use of metallic or other autoclavable material for anchors14,16, and possibly chamber12ensures a relatively simple cleaning process and allows for reuse of apparatus. In some embodiments, anchors14,16could be non-reusable parts and simply discarded after muscle tissue is removed during harvesting. The use of electromagnets29allows for easy and quick coupling and uncoupling, and holder36and cap28help to ensure the magnetic field does not affect the tissue growth. The use of fans34and metal sink38also ensure that the heat generated does not affect the growing conditions within chamber12.

FIG.2Adepicts an embodiment of a chamber12for use in apparatus10;FIG.2Bshows a cross-sectional view of the chamber12; andFIG.2Cshows a top view of the chamber12. Chamber12includes first end22, second end24, largest cross-sectional point25, inlet30, outlet32and cavity33. Cavity33at first end22can be a blind hole and/or shelf for receiving and properly positioning anchor14; and second end24is generally cylindrical in shape and open for cap28to cover or connect to and through.

Chamber dimensions include length L of 70-80 mm, radius RLat largest cross-section of about 15.5 mm, radius RFEat first end of 5 mm; top opening diameter TOD of 25 mm; outer diameter OD at thickest part of about 36 mm, and an outer wall thickness of 3 mm. These are example dimensions for a chamber of 250 mL, and dimensions would vary depending on the size of chamber required, the muscle tissue being produced, the material used for chamber, conditions for production of muscle, etc. Such a configuration could also be used for chambers from 25 mL up to 1-2 L or even more, for example 500 mL to 1 L. This allows form much larger chambers, and consequently larger portions of cultured tissue than prior art chambers.

Chamber12can be formed of glass, though could be a metal such as stainless steel, or other suitable materials. Chamber can be formed by additive manufacturing, casting and milling or other suitable manufacturing processes.

Chamber12is symmetric around its vertical axis, and shaped to have an expanding cross-section from each of its ends22,24. The cross-section is largest at middle point25, which is generally around a mid-point of chamber12between ends22,24, but does not have to be. In this embodiment, cross-sections are circular though they could be different shapes (e.g., oval) in other embodiments. The walls of chamber12shown inFIGS.2A-2Care curved, though other embodiments could have less (or more curvature), including embodiments with little to no curvature as that shown inFIGS.4A-4C.

The design of chamber12with an expanding cross-section from each of the ends22,24allows for production of muscle tissue with consistent shape between anchors. Prior art chambers typically had straight or parallel sides (e.g., rectangular or cylindrical), which resulted in growth of muscle tissue that had a larger cross-section at the ends, and a smaller cross-section in the middle, or concave sides with an hourglass shape in the tissue produced. It was surprisingly discovered that designing chamber12to have an expanding cross-section from each of the ends with a largest cross-section at a middle point resulted in muscle tissue that was substantially uniform or even convex between anchors14,16, thereby resulting in more muscle tissue growth (larger volume), and overall more efficient production. The shape was also more desirable for usage and for consumer preferences.

In some embodiments, the curvature of chamber12is different, for example, the chamber could expand even further to have a largest cross-section much larger than ends and anchors14,16. Such a configuration could be used to form muscle tissue with a convex shape, which more closely aligns with that from animals—closely mimicking muscle tissue which consumers expect from non-lab grown muscle tissue.

Chamber12wall can include one or more heating wires directly in the walls of chamber12to precisely regulate the temperature inside chamber12. Alternately or additionally, chamber could include an outer sleeve to help regulate temperature. Some embodiments could even include a fluid flow between the outer sleeve or jacket and the chamber12to help control temperature in an efficient and economical way. The growth of muscle tissue is a very temperature sensitive process, and certain parts of the procedure, such as the cooling down must be done very precisely to ensure that the texture of the muscle tissue does not degrade during the process. Such heating wires or other temperature regulators built directly into apparatus10can ensure that the temperature is accurately and precisely controlled.

FIG.3Ashows a perspective view of a three-dimensional anchor14; andFIG.3Bshows a cross-sectional view of anchor14. While anchor14is referenced, this could also be anchor16. Anchors14,16would typically have the same configuration, though could be different in some embodiments. Anchor14is in a biomimetic configuration, resembling a tendon from an animal. Example dimensions can include an outer diameter OD of 25 mm, length L of 2-10 mm, and a perimeter wall thickness PWT of 1 mm. Anchor14dimensions, particularly length, can vary depending on the volume of chamber and attachment strength required. The anchor14shown would be suitable for use with the apparatus ofFIGS.1A-1B,4A-4Cand/or the chamber ofFIGS.2A-2C.

Anchor14is a biomimetic shape, modelled after a projection of a tendon, and having a three-dimensional shape to which tissue can attach. Anchor14includes perimeter34and a plurality of attachment paths36. Attachment paths36form various three-dimensional pathways throughout the interior of perimeter34. In the embodiment shown, perimeter34is cylindrical, but could be another shape in other embodiments. Perimeter34typically has a solid outer wall extending longitudinally, with the paths36forming winding interconnected pores through the longitudinal direction within perimeter34. Attachment paths36do not typically extend the length of perimeter, and instead different paths36start at different points along the inner circumference of perimeter34, and do not generally align in the vertical direction, though in some embodiments, they could align. The embodiment shown has a sort of wheel and spoke pattern, with an open inner hub40and spokes42extending from the inner hub40to the outer perimeter34. Such a configuration can be useful for a connector rod26(seeFIG.1A) to extend into anchor14inside of inner hub40, thereby securely connecting to anchor14. In some embodiments, the same pattern shown in the cross-section ofFIG.3Bcould be simply repeated and rotated along the vertical axis of anchor14inside perimeter34to form the plurality of attachment paths36connecting at various three-dimensional points with each other and with the perimeter34.

Anchor14can be formed of a conductive material, for example metals or alloys, e.g., Titanium. Anchor14can be formed by casting, machining, or other methods depending on the specific design, materials and size. Using a conductive material allows for connection to one or more electrodes to provide direct electrical stimulation to chamber12and muscle tissue for more efficient compaction and growth.

Cross-sectional view of anchor14shown inFIG.3Balso shows example attachment points38for muscle tissue to pathways36. As can be seen from this cross-sectional view, the plurality of pathways36result in many points38for the muscle tissue to connect or attach to anchors14, both along the perimeter34and along all sides of the pathways36. The three-dimensional configuration allows for different attachment points38in different planes, thereby giving the muscle tissue stronger connections to anchors14,16in the x, y and z directions.

As mentioned above, mechanical stimulation helps in the efficient growth and development of muscle tissue. The three-dimensional configuration of anchor14with a large number of attachment points38in all directions allows for mechanical stimulation to put tension on the muscle tissue in all directions (x, y and z) through various movements, while maintaining a reduced risk of muscle tissue detaching from anchor14. With such a configuration, mechanical stimulation can be performed (e.g., rotational and/or vertical movements) which would result in strain of up to 0.2-0.3, both in the longitudinal and rotational direction. In the chambers shown, this would be about 1 mm of movement, though the movement amounts would vary depending on the muscle tissue being grown, anchor design, size of chamber, etc. Past systems could only perform mild mechanical stimulation due to the risk of detachment of muscle tissue from anchors. By using the three-dimensional biomimetic anchors14,16where muscle tissue wraps itself around and through to attach at a large number of points on all sides of attachment paths36and inside of perimeter34of anchor14, apparatus10is able to produce larger volumes of muscle tissue more efficiently. Anchors14,16are also able to provide a large number of attachment points38in a compact volume, thereby allowing for easy removal from one end of chamber12(through top opening) when muscle tissue has matured.

FIG.4Ashows a perspective view of a second embodiment of apparatus10′ for culturing muscle tissue;FIG.4Bshows a side view of that apparatus10′; andFIG.4Cshows an exploded view of apparatus10′. Similar parts are labeled similarly to those inFIGS.1A-3B.

Apparatus10′ works in the same manner as apparatus10described in relation toFIGS.1-3B. However, in this embodiment, chamber12′ is generally cylindrical from the first end to the second end. This can be useful for culturing muscle tissue which compacts very little during maturation, allowing for growing a generally cylindrical muscle tissue between anchors14,16.

Each of anchors14,16is metallic and secures to electromagnets29for positioning within chamber12′ for culturing muscle tissue. Metal sink38acts as a heat sink, and fan34helps to blow the heat away so it does not affect the chamber12′ temperature or the muscle tissue culturing. Cap28can also have a fan inside, which is not shown in the Figures. Holder36helps to position anchor14at a sufficient distance from electromagnet29so that the magnetic field generated does not affect the muscle tissue. Cap28can perform a similar function.

The electromagnetic coupling of anchors14,16allows for secure positioning of anchors14,16and therefore tissue within chamber12′, and for easy and quick uncoupling when it is time to harvest the tissue. For culturing tissue, anchor14is placed on holder36, with cap28holding anchor16placed on top of chamber12′. Electromagnets29are then activated, and a magnetic field is produced, which holds each of anchor14and16in place, respectively. When the tissue has been cultivated (as described above), electromagnets29are deactivated and cap28with anchor16can be moved vertically upwards out of chamber12′. This can be manually or through a machine, arm, or other device. The grown tissue connected at one end to anchor16and at the other end to anchor14moves with anchor16(also bringing anchor14) out of the top of chamber12′. Once removed, tissue can be removed from anchors14,16, e.g., by cutting.

Chamber12′ with anchors14,16and electromagnetic couplings provides a system where relatively large amounts of muscle tissue can be grown and easily removed when maturation has taken place. The use of electromagnets29allows for easy and quick coupling and uncoupling, and holder36and cap28help to ensure the magnetic field does not affect the tissue growth. The use of fans34and metal sink38also ensure that the heat generated does not affect the growing conditions within chamber12′. Cylindrical chamber12′ is particularly useful when growing tissue which experiences little to no compaction.

FIG.5Ashows a view of a shower cap50, which could be used with apparatus10,10′ as an alternative way to introduce liquid and/or media into chamber12,12′.FIG.5Bshows a cross-sectional view of shower cap50.

Shower cap50includes inlet52, channel54and outlets56. Generally the shower cap would connect to chamber12,12′ at the upper end either inside of or in place of cap28. Anchor16would fit through the center of shower cap50and could be moveable up and down with respect to shower cap, as described above.

Media would enter into shower cap50through inlet52and flow into annular channel54. Outlets56, which could be holes, microtubes or other outlet configurations, are equally spaced around channel54and around a central axis of the chamber. Thus, the outlets56are generally located around the circumference or perimeter of the anchor below and the eventual tissue. The media flows out of outlets56into chamber12,12′.

The equidistant spacing and plurality of outlets56located all around the shower cap50provide an inflow of media all around top anchor16and tissue, thereby providing for better flow and distribution of media all around the tissue and chamber. This leads to better mixing in the chamber as well as better perfusion. Typically shower cap50would eliminate side inlet30shown in chambers12,12′ being used in place of that media inlet. However, in some embodiments, shower cap50could be used in addition to inlet30.

FIG.6Ashows a perspective view of an alternative anchor60;FIG.6Bshows a side view of anchor60;FIG.6Cshows a top view of anchor60. Anchor60is an alternative to anchors14,16depicted and described above. Anchors60can be formed of a metallic material, or other suitable materials.

Anchor60is an auxetic design, with the building blocks62shown inFIGS.7A,7B and7C. The building blocks62shown are butterfly shaped hexagon prisms connected at a center point, which can deform into square or rectangular shapes when an outward force is applied. The building blocks62are repeated in the x-direction, where left vertical bar of the right block also functions as the right vertical bar of the left block. In the y-direction, the blocks62are simply stacked. In the embodiment shown, anchor60extends two building blocks62in the vertical direction, and is a square shape, extending six building blocks62on each side, as seen inFIGS.6B and6c. Other embodiments could have a different shape, for example, circular or another polygonal shape and/or a different configuration.

Example dimensions can be HA of about 4.9 mm; WBB of about 4.0 mm; and a side length SAof about 10.87 mm. These are example dimensions only, and can vary depending on chamber and tissue requirements.

FIGS.6D and6Eshow schematic depictions of how anchor60behaves, withFIG.6Dshowing anchor60in a relaxed state; andFIG.6Eshows anchor60in a stretched state. The auxetic design of anchor60means that when stretched or elongated in one direction, the anchor becomes thicker perpendicular to the applied force. Thus, the relaxed state, as shown inFIG.6Dis more compact than when forces, such as tissue growth and/or mechanical stimulation put forces on anchor. Force Fr could represent the force of the tissue pulling anchor60upward in a vertical direction, with force FcR representing the force of the connector rod holding anchor60in place within the chamber. These two forces acting on anchor60result in the horizontal forces seen elongating the anchor in that direction, perpendicular to the forces applied.

FIG.6Fshows anchor60in use in a chamber12′; andFIG.6Gshows a top view of anchor60in use in chamber12′. Chamber12′ is a schematic depiction and does not include all parts which chamber12′ would typically have (as is shown in FIGS.1A2C and4A-4C). Anchor60is shown at the lower end of chamber12′, connected through connecting rod60, similar to anchors14shown and described in the previous figures. Chamber12′ would also include a top anchor60, extending from a connecting rod from the upper end of the chamber12′. Tissue would connect and grow between the upper and lower anchors60.

Anchor60, by forming from a plurality of building blocks62in an auxetic design, provides a large amount of places for tissue to wrap around and to which to connect. The use of relatively small building blocks providing small pores has been shown to result in a positive influence in the attachment of the tissue to anchor60. The auxetic design allows for the use of these small building blocks62in a repeated manner to build up anchor60to any size required. Due to the specific design of the building blocks62and anchor60, the attachment points can be located on a number of sides, both in the interior and on the outside of the anchor60. This results in very strong connections between the anchor60and tissue, allowing for the growth of larger muscle tissue and for more mechanical stimulation, resulting in more efficient growth and muscle tissue quality in terms of texture, appearance and taste. Additionally, the auxetic design provides for additional strength, even when subject to the mechanical stimulation loads, with forces in one direction resulting in elongation in another. The use of a metallic or conductive material to form anchor60allows for the direct connection and/or integration of one or more electrodes into the apparatus to provide electrical stimulation to chamber, to promote cell differentiation and tissue compaction as well as muscle tissue growth systems and components shown and discussed in relation toFIGS.1A-2Ccan be used with apparatus10′ and chamber12′ ofFIGS.4A-4C, and vice versa. Systems and components shown and described inFIGS.5A-7Ccould also be used with any of the chambers and apparatuses shown.