Harvest friendly single-use cultured meat bioreactor

Abstract

The present invention contemplates single-use cell culture bioreactors suitable for the production of cultured meat products where the bioreactor comprises sections designed to be separated for harvesting the cultured meat product with minimal or no damage to the product; nor loss of biomass during harvesting.

Description

Attorney Docket No.: P24-154-SEC-WO01HARVEST FRIENDLY SINGLE-USE CULTURED MEAT BIOREACTORCross-Reference to Related Applications

[0001] The present application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 687,647, filed August 27, 2024, the entire content of which is incorporated herein by reference.Background

[0002] Lab grown meat or meat-like products, often referred to as “clean meat,” “cultivated meat” or “cultured meat,” are likely to be a significant player in providing food for the ever-increasing human population. However, efforts in this field have only met with limited success.

[0003] In order to generate structured cultivated meat, scaffolding must be used. See, e.g., International Patent Publication No. WO 2002 / 038240 A2 to Merck Patent GMBH. To be practicable and cost effective, the scaffolding must be edible and / or dissolvable and result in a texture and structure in the final product that gives a mouth feel reminiscent of real meat or natural meat ( / .e., meat derived from an animal). This means that the scaffolding must provide for at least three qualities: 1) be edible, 2) provide a texture and mouth feel similar to real meat and, 3) be a suitable culture environment for myocytes or myocyte-like cells and other cell types to grow efficiently and form muscular structure (for example, form myotubules and achieve tissuelike cell densities) resembling natural muscle.

[0004] However, creating a cultured meat product that resembles a natural meat product for taste, texture, structure, cookability and mouth feel is only part of the solution. Part of the reason is because the cultured meat product generated with current bioreactor technology is difficult to harvest cleanly and without disrupting or damaging product. Even with success of production of a structured meat product, harvesting the product such that the structure of the meat product remains intact is another problem faced by the industry. This is, in part, because effective culture apparatus that allow for the non-disruptive harvesting of the meat product are not available. Current methods of harvesting a cultured meat product can best be described as “man handling” and the result is what would be expected by using such techniques. Any structure to the product is partially or totally lost. The industry faces a sizableAttorney Docket No.: P24-154-SEC-WO01 problem in being able to both grow and then successfully harvest a culture meat product. What is needed are bioreactors that provide for easy harvesting of the culture meat product without causing damage to the cultured meat product.Summary of the Invention

[0005] The present invention solves the problem of damage caused in harvesting a structured cultured meat product. The present invention allows for the harvest of a cultured meat product with no damage or only minimal damage to the product. The present invention is directed toward a single-use cell culture bioreactor (the ’’bioreactor”) that can be easily separated from the cultured meat product. The bioreactor of the present invention is made of a hard, flexible or semiflexible plastic or plastics ( / .e., one or more plastics). The bioreactor comprises a housing having a housing wall, the wall enclosing a lumen. The housing wall has “failure points” built in where sections of the housing wall can be separated from each other either totally or partially. This allows the removal of the housing from the culture meat product inside ( / .e., in the housing lumen) with no or minimal damage to the cultured meat product.

[0006] In one embodiment, the housing is cylindrical ( / .e., a tube) having a length and a diameter. In one embodiment, the “failure points” are grooves along the outer surface of the housing wall. In a preferred embodiment, the grooves are cut or molded in the outer surface of the housing wall. The grooves may run partially or totally along the length of the housing and / or encircle the diameter of the housing. While the bioreactor is described herein as a tube, the tube need not be round. It can be any useful or desired cross- sectional shape. Likewise, the tube, while generally elongated, is not limited to any dimensional configuration. That is, it is not limited to specific aspect ratios with regard to length and diameter. One of skill in the art, armed with the teachings of this specification, will be able to determine the dimensions of the bioreactor of the present invention suitable for a particular use without undue experimentation.

[0007] The bioreactor housing of the present invention may have one or more ports for providing media to the cell culture and to allow for the removal of waste products. The bioreactor housing also has a first end and a secondAttorney Docket No.: P24-154-SEC-WO01 end, the first end and / or the second end may be open or closed depending of desired usage.

[0008] Fittings may be used to connect the bioreactor of the present invention to tubing or other means for providing media and gases to the lumen of the bioreactor and take waste products away. The tubing may be connected to the port or ports, if present, or to the endcaps.

[0009] Harvesting of the bioreactor of the present invention involves peeling or causing to be peeled the sections of the housing along the grooves. This can first involve snapping off the end(s) of the bioreactor of the present invention at the groove(s) (“circumferential grooves”) that encircle(s) the housing and then peeling one of more sections from the housing along the grooves that run along the length (“longitudinal grooves”) of the housing to expose the cultured meat product. Once exposed, the cultured meat product can be easily separated from the reminder of the housing.

[0010] The present invention contemplates a single use cell culture bioreactor housing, comprising: an elongated tubular element, the elongated tubular element having a first end and a second end, a wall defining a lumen and having an inside surface and an outside surface, the wall comprising at least two wall elements that are at least partially separable from each other along longitudinal separation lines running the length or substantially the length of the elongated tubular element; at least one fluid path through the bioreactor housing for carrying culture media into the bioreactor housing and spent media out of the bioreactor housing.

[0011] The present invention further contemplates that the elongated tubular element further comprises at least two longitudinal grooves at the longitudinal separation lines extending the length of the elongated tubular element or substantially the length of the elongated tubular element on the outside surface of the wall, the longitudinal grooves reducing the thickness of the wall by at least 50% as compared to the non-grooved wall of the housing.

[0012] The present invention still further contemplates that the wall elements of the elongated tubular element are held together with snap-fit joints located at the longitudinal separation lines.

[0013] The present invention still further contemplates that the cell culture bioreactor housing of further comprises a scaffolding matrix located in theAttorney Docket No.: P24-154-SEC-WO01 lumen of the bioreactor housing, the scaffolding matrix suitable for the culture of attachment dependent cells such as myocytes.

[0014] The present invention still further contemplates that the scaffolding matrix fills at least 30%, at least 40%, at least 50% or at least 60% of the lumen.

[0015] The present invention still further contemplates that the scaffolding matrix comprises a plurality of hollow fibers.

[0016] The present invention still further contemplates that the scaffolding is edible.

[0017] The present invention still further contemplates that when the scaffolding matrix comprises hollow fibers, the hollow fibers extend from the first end to the second end of the elongated element of the housing.

[0018] The present invention still further contemplates that the cell culture bioreactor housing further comprises a circumferential groove on the outer surface of the wall proximal to the first and / or the second ends and intersecting with the longitudinal grooves.

[0019] The present invention still further contemplates that the cell culture bioreactor housing further comprises removable endcaps attachable to and detachable from the first and / or second ends of the elongated tubular element.

[0020] The present invention still further contemplates that the cross-sectional shape of the elongated tubular element of the cell culture bioreactor housing of the present invention is selected from round, oval, ellipsoid, square and rectangular.

[0021] The present invention still further contemplates that the number of longitudinal lines of the cell culture bioreactor housing of the present invention are from two to ten.

[0022] The present invention still further contemplates that the width of the elongated tubular element of the cell culture bioreactor housing of the present invention is from 3 mm to 30 cm.

[0023] The present invention still further contemplates that the length of the elongated tubular element of the cell culture bioreactor housing of the present invention is from 6 cm to 3 meters.Attorney Docket No.: P24-154-SEC-WO01

[0024] The present invention still further contemplates that the longitudinal and circumferential grooves of the elongated tubular element leave about 0.2 mm to about 1 .0 mm of the wall thickness in the elongated tubular element or about 0.4 mm of the wall thickness in the elongated tubular element.

[0025] The present invention still further contemplates that the longitudinal and circumferential grooves delineate bioreactor housing wall elements of the elongated tubular element and provide for a failure area or areas that allow for the at least partial separation of two or more of the wall elements from each other.

[0026] The present invention still further contemplates that the cell culture bioreactor housing of the present invention, wherein the wall elements are completely separatable from each other.

[0027] The present invention still further contemplates that the cell culture bioreactor housing is opaque, transparent or translucent.

[0028] The present invention still further contemplates that the cell culture bioreactor housing is flexible, essentially rigid or rigid.

[0029] The present invention still further contemplates that the cell culture bioreactor housing is biodegradable.

[0030] The present invention still further contemplates the cell culture bioreactor housing of the present invention, where the elongated tubular element comprises one, two or more ports in the wall to provide for a fluid path.

[0031] The present invention still further contemplates the cell culture bioreactor housing, wherein, upon harvesting, the ports are used to provide leverage for initiating the separation of the bioreactor housing wall elements.

[0032] The present invention still further contemplates the cell culture bioreactor housing of the present invention, wherein the elongated tubular element is molded from one or more plastics.

[0033] The present invention still further contemplates the cell culture bioreactor housing of the present invention, wherein the elongated tubular element is made of two or more bonded sections.

[0034] The present invention still further contemplates the cell culture bioreactor housing of the present invention, wherein the bioreactor is a singleuse bioreactor.Attorney Docket No.: P24-154-SEC-WO01

[0035] The present invention still further contemplates the cell culture bioreactor of the present invention, wherein the longitudinal and circumferential grooves are molded, extruded, cut or both.

[0036] The present invention contemplates a single use cell culture bioreactor housing, comprising: an elongated tubular element, the elongated tubular element having at least a first end and a second end, a wall having an inside surface and an outside surface, the wall defining a lumen, and the wall comprising at least two wall elements; at least one fluid path through the bioreactor housing for carrying culture media into the bioreactor housing and spent media out of the bioreactor housing; wherein the wall elements are reversibly attachable to each other and are held together with snap-fit joints.

[0037] The present invention contemplates a single use cell culture bioreactor housing, comprising: an elongated tubular element, the elongated tubular element having a first end and a second end, a wall having an inside surface and an outside surface, the wall defining a lumen; at least one fluid path through the bioreactor housing for carrying culture media into the bioreactor housing and spent media out of the bioreactor housing; wherein the bioreactor housing further comprises at least two longitudinal grooves extending the length of the elongated tubular element or substantially the length of the elongated tubular element on the outside surface of the wall, the longitudinal grooves reducing the thickness of the wall by at least 50% as compared to the non-grooved wall of the housing.

[0038] The present invention contemplates that a method of culturing and harvesting a cell culture product, the method comprising: providing; a single use bioreactor housing of any of the present invention; seeding interior space of the single use bioreactor with one or more cell types; culturing the cells by providing culture media to the cells and removing spent media from the cells via the fluid path until the cell culture reaches about 75% confluency or greater to create a structured cell culture product; stopping the flow of culture media into and the flow of spent media out of the bioreactor; separating, at least partially, one wall element elongated tubular element from another wall element from the structured cell culture product thereby permitting the removal of the structured cell cultured product from the bioreactor housing.Attorney Docket No.: P24-154-SEC-WO01

[0039] The present invention still further contemplates a method of culturing and harvesting a cell culture product, the method comprising: providing; a single use cell culture bioreactor having an elongated tubular element, the elongated element having 1) a first end and a second end, a wall having an inside surface and an outside surface and a lumen; 2) at least one fluid path through the bioreactor housing for carrying culture media into the bioreactor and spent media out of the bioreactor housing; 3) wherein the bioreactor housing further comprises at least two wall elements that are at least partially separable from each other along two or more longitudinal separation lines between the first and the second ends, and; 4) a scaffolding material in the interior space of the elongated tubular element; seeding interior space of the single use bioreactor with one or more cell types; culturing the cells by providing culture media to the cells and removing spent media from the cells via the fluid path until the cell culture reaches about 75% confluency or greater to create a structured cell culture product; stopping the flow of culture media into and the flow of spent media out of the bioreactor; separating, at least partially, one wall element elongated tubular element from another wall element from the structured cell culture product thereby permitting the removal of the structured cell cultured product from the bioreactor housing.Brief Description of the Figures

[0040] Fig. 1 shows a perspective view of one embodiment of the single-use cell culture bioreactor present invention.

[0041] Figs. 2A- 2D show: (A) a schematic of a circumferential cross-section of one embodiment of the single-use bioreactor of the present invention at a port; (B) a longitudinal cross-section of the single-use cell culture bioreactor present invention; and (C) a longitudinal cross-section of the end of the singleuse cell culture bioreactor of the present invention showing a circumferential groove and a port fluidly connected to the lumen of the bioreactor and (D) showing a cross section of the elongated tubular element showing the longitudinal grooves.

[0042] Figs. 3A & 3B show (A) a schematic representation of an embodiment of the single-use cell culture bioreactor of the present invention. The magnified insert (B) shows the intersection of a longitudinal groove and a circumferential groove.Attorney Docket No.: P24-154-SEC-WO01

[0043] Figs. 4A & 4B show (A) a rigid version of the single-use cell culture bioreactor with (B) the endcaps snapped off in the second picture.

[0044] Figs. 5A- 5C show (A) a flexible version of the single-use cell culture bioreactor with (B) a wall section partially removed and (C) a wall section completely removed..

[0045] Fig. 6 shows hollow fibers after harvesting by cutting the endcap off of the bioreactor. Shards of plastic are clearly visible on the end of the hollow fiber bundle showing that this is not a desired harvest method.

[0046] Fig. 7 shows micrographs of cells growing on the hollow fibers from a single-use bioreactor of the present invention after harvesting by separating the bioreactor by peeling away housing sections using the failure points denoted by the longitudinal and circumferential grooves.

[0047] Figs, 8A & 8B show (A) a rigid bioreactor housing of the present invention prior to insertion of a hollow fiber bundle and (B) a flexible bioreactor housing of the present invention prior to the insertion of a hollow fiber bundle.

[0048] Figs. 9A- 9C show (A) a bioreactor housing of the present invention held as if a user was going to “snap” open the housing with the testing device in the background, (B) a bioreactor hosing of the present invention set up in the testing device and (C) the bioreactor housing of the present invention undergoing testing to determine the level of force necessary to “snap” off one of the ends of the bioreactor housing.

[0049] Fig. 10 shows a line graph of breaking force in pounds-force vs. travel in millimeters of the impact generating arm of the device. The test was repeated at least five times.Detailed Description of the Invention

[0050] Definitions

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] When introducing elements of the present disclosure or the preferred embodiments(s) thereof, the articles "a," "an," "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.Attorney Docket No.: P24-154-SEC-WO01

[0053] The transitional phrases “comprising,” “consisting essentially of” and “consisting of” have the meanings as given in MPEP 2111 .03 (Manual of Patent Examining Procedure; United States Patent and Trademark Office, 9thEd., Revision Feb 2023 [R-07.2022]). Any claims using the transitional phrase “consisting essentially of” will be understood as reciting only essential elements ( / .e., the basic and novel characteristics) of the invention and any other elements recited in dependent claims depending from the claim using the transitional phrase “consisting essentially of” are understood to be non- essential to the invention recited in the claim from which they depend.Likewise, any additional elements over those claimed that are described in a prior art reference(s) are excluded from the claim by use of the transitional phrase “consisting essentially of.”

[0054] Any numerical ranges provided herein include all numbers within the range as though they had been specifically recited. Thus, for example, a range of 10 - 100 also includes each and every number between 10 and 100 including without exception every whole number and every fractional or decimal number between the recited numbers. Further, phrases such as “at least” and “no more than” are understood to recite ranges starting with or ending with the recited number, respectively, and include all numbers within that range as though they had been specifically recited.

[0055] The terms “single-use cell culture bioreactor,” bioreactor,” “reactor,” single-use reactor,” etc., when use to refer to the single-use cell culture bioreactor present invention, are considered to be synonymous and may be used interchangeably herein.

[0056] A “structured meat product” or “structured cultured meat product” is a cell culture grown meat product having a texture and structure like, similar to or suggestive of natural meat from animals. The structured meat product of the present invention has a texture and structure that resembles natural meat 1) in texture and appearance, 2) in handleability when being prepared for cooking and consumption (e.g., when being sliced, ground, cooked, etc.) and 3) in mouth feel when consumed by a person. The materials and methods of the present invention, when used in the production of structured clean meat, achieve at least one of these criteria, two of these criteria or all three of these criteria. The terms “clean meat,” “clean meat product,” “cultured meat,Attorney Docket No.: P24-154-SEC-WO01“cultured meat product,” and “structured cultured meat product” when used herein, shall be understood to be synonymous and interchangeable and shall be understood to refer to a structured cultured meat product as defined herein.

[0057] The terms “snap-fit” or “snap-in” joints, as used herein, refers to assembly components and methods known to those of skill in the art to attach flexible parts. The snap-fit is reversible as is evident by its use in, for example, removable and replaceable battery compartment covers. There are a number of variations in snap-fits, including cantilever, torsional and annular. See, for example, en.wikipedia.org / wiki / Snap-fit; info.rapidaxis.com / blog / snap-fits-what-they-are-how-they-work-and-how-to- use-them; US 3,596,940 to Horwitt; and, Snap-Fit Joints for Plastics, Bayer Material Science. It is contemplated that devises made with snap-fit wall sections may be reusable for some applications.

[0058] The term “bonded,” as used herein, refers to wall elements that are adhered to each other by, for example, heat or adhesive. Bonded wall elements of the present invention will still have the function of being separatable from each other, i.e., they will be design to function as longitudinal separation lines.

[0059] The term “longitudinal lines” or “longitudinal separation lines” as used herein, refers to areas on the wall of the elongated tubular element that run the length or run substantially the length of the elongated tubular element and are designed for partial or total separation of one wall element from another wall element. Non-limiting examples of longitudinal separation lines are grooves, cuts, snap-fit joints and materials of a differing strength (e.g., weaker) than the wall elements that allow for easy separation of the “wall elements” (also referred to herein as “housing wall elements”) when a force of suitable strength and direction is applied (see, e.g., Example 3).Single-use Cell Culture Bioreactor

[0060] The present invention contemplates a bioreactor for the culture of an edible structured cultured meat product that permits the non-destructive harvesting of the product. In the context of the present invention, the term “non-destructive” refers to a process wherein the cultured meat product is not damaged or only minimally damaged during harvesting from the bioreactor inAttorney Docket No.: P24-154-SEC-WO01 which it was grown. “Minimally damaged” refers to less than 25%, less than 20%, less than 15%, less than 10%, less than 8%, less than 6%, less than 4%, less than, 2%, less than 1 % or less than 0.5% of the cultured meat product has been damage. “Damage” is defined herein as having the structural integrity of the cultured meat product disrupted such that the structural integrity is lessened to the degree that the structure of the cultured meat product is no longer visually recognizable. It is understood that a minimal amount (up to about 2%, 4%, 6% 8% or 10% of the total) of the cultured meat product in the bioreactor may be unincorporated into the structure of the cultured meat product due to variations in the individual cultures and individual culture environments and that this variation is not to be considered “damaged” contributable to the harvesting process but, rather, a normal biproduct or result of the structured cultured meat production process.

[0061] It is also understood that harvesting of prior art cultured meat bioreactors typically causes a loss of product through the harvest process. Although not “damaged” as defined above, loss of the cultured meat biomass through, for example, separation of portions of the biomass from the encased product resulting from the harvest process is a problem within the industry. It is estimated that as much as 30 - 40% of the product may be lost in this way. Without a user friendly method of harvest, this problem will continue in the industry.

[0062] It is contemplated that the harvest friendly bioreactors of the present invention are single use, disposable bioreactors. They comprise a housing, one or more inlet ports and one or more outlet ports. The housing comprises an inner wall and an outer wall. The area within the housing is the lumen of the housing, or “lumen.” The lumen is where structured cultured meat product is produced.

[0063] It is contemplated that the inlet port(s) are useful and suitable, for example, for providing culture media to the cells seeded and growing in the bioreactor of the present invention. The outlet port(s) are useful and suitable, for example, for the elimination of waste products including spent cell culture media and cell waste products from the bioreactor of the present invention.

[0064] It is contemplated that the bioreactors of the present invention are made from one or more plastics. The one or more plastics may have a degreeAttorney Docket No.: P24-154-SEC-WO01 of flexibility such that they impart a degree of flexibility on the wall(s) of the bioreactor of the present invention. In another aspect, the plastic may be rigid. In another aspect of the present invention, portions of the bioreactor may be flexible and other portions of the bioreactor may be rigid. For example, the housing may be flexible and the endcaps may be rigid. In another example, the housing may be made of two or more plastics where, for example, the housing is made of both rigid plastic with flexible, peal-away sections. The plastics are also moldable and can be shaped after molding if needed or desired by, for example, scoring or cutting.

[0065] Examples of suitable plastics include, but are not limited to, polystyrene, acrylonitrile butadiene styrene (ABS), cyclic olefin copolymer (COC), polycarbonate, polylactic acid (PLA), Polybutylene adipate terephthalate (PBAT), or other glassy / amorphous polymers that can be fractured easily at room temperature. Typically, a polymer useful in the present invention has a glass transition temperature (Tg) below room temperature. Suitable plastics, when the bioreactor of the present invention is used for food production, are materials known to be safe for food contact as determined by, for example, the Food and Drug Administration (FDA) or materials that are generally recognized as safe (GRAS). One of skill in the art, in combination with the teachings of this application, can identify further plastics or other materials suitable for use in the bioreactors of the present invention without undue experimentation. Further, biodegradable polymers are suitable and preferred for use as a material for the housing of the bioreactor of the present invention.

[0066] The single-use cell culture bioreactors of the present invention may be molded from a suitable plastic or plastics. In other cases, the bioreactor may be formed by extrusion or by machining, for example, from a block or tube made of one or more suitable plastics. Extruded bioreactors may be made with one layer of material or with multiple (two or more laminated layers) layers of material. For example, a flexible layer may form the internal wall of the bioreactor and another layer(s) may form an outer layer to provide support to the structure. The outer layer may be more ridged than the inner layer. The outer layer would be discontinuous leaving spaces between the discontinuous sections of the outer layer, i.e., it would provide for theAttorney Docket No.: P24-154-SEC-WO01 longitudinal and / or circumferential grooves. This would leave the outer surface of the flexible inner layer exposed to the environment where it was not covered with the more outer layer and provide for an easy “break-away” point for opening the bioreactor and harvesting the cultured meat product.

[0067] Thus, the bioreactors of the present invention, comprise a housing, said housing, in one embodiment, being elongated with the length of the housing being a greater dimension than the diameter of the housing. The housing can, for example, be 4 times, 5 times, 6, times, 7 times, 8 times, 9 tines, 10 times, 12 times, 15 times, 20 times longer or more than the diameter of the housing. The housing has a wall with an inside surface and an outside surface, said wall delineating a lumen. One or both ends of the housing may be open or closed. The cross-sectional shape of the housing may be round, oval, ellipsoid, rectangular, square or any other shape that is desired and usable.

[0068] In one aspect, the bioreactor of the present invention may have one or more ports that are fluidly connected to lumen of the housing, said ports may be 90 degrees ( / .e., perpendicular) to the longitudinal direction ( / .e., the length) of the housing or any other angle that is desired and useful. The number of ports is not limited and may be determined by the needs of the culture conditions required and / or the size of the culture vessel with larger vessels needing more inlet and outlet ports. In some aspects unused ports may be closed or sealed if not needed. The ports may be made to be used with, for example, a sanitary fitting (Sanitary Fittings, LLC Chicago, IL) , a Luer® Lok (Becton Dickinson, Franklin Lakes, NJ) fitting, a hose-barb fitting, a threaded screw fitting, a quick connect (such as a Swagelok fitting (Solon, OH)) or a sterile connector (such as Lynx fittings (MiliporeSigma, Burlington, MA)).

[0069] In another aspect, the endcaps of the bioreactor of the present invention may be open ( / .e., forming another port or ports, if one of both of the endcaps are open, respectively) or closed. Further, the endcaps may be separatable from the housing. That is, for example, push on / pull off ( / .e., a friction fit) or screw on / off. The one or both of the endcaps may be sealed, i.e., closed, or open, / .e., forming a port. As with the ports on the housing (see, previous paragraph), the endcaps may be made to be used with, forAttorney Docket No.: P24-154-SEC-WO01 example, a TC fitting, a Luer® Lok fitting, a hose-barb fitting, a threaded screw fitting, a quick connect (such as a Swagelok fitting (Solon, OH)) or a sterile connector (such as Lynx fittings (MiliporeSigma, Burlington, MA)).

[0070] The endcaps may be molded onto the housing or may be made separately from the housing. If the endcaps are separate from the housing they may be assembled with the housing by friction fit ( / .e., a push fit) or may be screwed onto the housing. Different size bioreactors may require or be better suited to one style of endcap or the other. For example, a larger bioreactor of the present invention may be better suited to or require screw-on endcaps or molded on endcaps due to the higher pressures the larger bioreactor is likely to be subject to when in use.

[0071] The single-use cell culture bioreactor of the present invention is not limited by size. Exemplary sizes are 3 ml, 300 ml, 3 L, 30 L, 300 L or greater. Other sizes are also envisioned. One of ordinary skill in the art will be able to choose a suitable reactor size for their particular needs with the assistance of the teachings of this specification.

[0072] The housing of the bioreactor of the present invention comprises one or more, two or more, three or more, four or more grooves scored, cut or molded into the housing wall the length of the housing or up to about 75%, 80%, 85%, 90%, 95% or 98% of the length of the housing. The grooves along the length of the housing may be referred to as “longitudinal grooves.” The grooves may extend to the first end of the housing, the second end of the housing or both the first and second end of the housing. The grooves that do not extend to the first end and / or the second end of the housing may, rather, end in a “circumferential groove” that, by definition, circles the diameter of the housing. In an alternative design, the longitudinal grooves may intersect and cross through the circumferential groove(s). The grooves (longitudinal and circumferential) are designed to be “failure points” where sections of the bioreactor housing can be separated partially or totally from each other. That is, they are engineered points of failure. Further, the areas between the grooves are referred to as bioreactor housing “sections” or “wall sections.”

[0073] The grooves of the housing may be any depth so long as the housing retains sufficient integrity to be used as a bioreactor and the groove can still function as an engineered point of failure when desired or needed, forAttorney Docket No.: P24-154-SEC-WO01 example, to facilitate harvesting. In one design of the housing, the grooves are cut into a depth of about 10%, 25%, 50%, 75% or 90% of the thickness of the wall of the housing. In another design of the housing, the portion of the housing wall retained after cutting the grooves into the housing is a thickness of about 10%, 25%, 50%, 75% or 90% of the thickness of the wall. The desired depth of the groove will depend upon the material or materials used for the housing, the overall thickness of the housing wall, the size of the bioreactor, the pressure used to deliver culture fluid (e.g., cell culture media) or other fluids (e.g., sanitization fluids such as acids and / or bases) to the bioreactor and the mechanism used to open or “peel” the bioreactor from the cultured meat product contained therein, e.g. manual removal or machinebased or machine aided removal of the housing. In one design, the thickness of the wall of the housing where the groove or grooves are cut is from about 0.2 mm -2.0 mm, from 0.3 mm to 1.0 mm, from 0.4 mm to 0.6 mm. In a preferred design, the wall thickness of the housing where the grooves are cut is about 0.4 mm. In another design, the wall of the housing where the grooves are cut is no more than 2.0 mm, 1 .0 mm, 0.8, no more than 0.6 mm, no more than 0.5 mm, no more than 0.4 mm, no more than 0.3 mm or no more than 0.2 mm. In another design, the wall of the housing where the grooves are cut are at least 0.2 mm, 0.3 mm, 0.4 mm, 0.5mm, 0.6mm, 0.8 mm, 1 .0 mm or 2.0 mm. One of ordinary skill in the art will be able to determine the depth that the grooves will need to be cut or molded (or otherwise formed) and the thickness of the wall at the groove, based on the teachings of this specification without undue experimentation. Further, the grooves may be from 1 mm to 10 mm wide (e.g., 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm or 10 mm wide), the with depending on at least the size of the bioreactor and the material used with larger bioreactors and bioreactors made from stiffer material requiring wider grooves to permit ease of removal of housing sections. One of skill in the art, armed with the teachings of this specification, will be able to determine a satisfactory width or widths for the grooves without undue experimentation. Preferably , the longitudinal grooves are evenly spaced but need not be. For example, when used with a larger bioreactor, pairs of grooves may be more closely spaced so as to provide a wall section that can be easily removed from the bioreactorAttorney Docket No.: P24-154-SEC-WO01 wall, much like a zipper or tear strip. If two such pairs of closely spaced grooves were opposite or approximately opposite each other on the circumference of the bioreactor housing, upon separation and removal of the two sections, the remaining wall sections could then be lifted off of the cultured meat product in the lumen of the bioreactor housing.

[0074] The bioreactor of the present invention is designed to be suitable for the production of a structured meat product, i.e., a cultured meat product. While the present invention is not limited to the nature of the material giving structure to the structured meat product, it is contemplated that the material comprises edible hollow fibers. See, for example, International Patent Publication No. WO 2002 / 038240 A2 to Merck Patent GMBH, which is incorporated herein by reference in its entirety, which describes the production and uses of edible hollow fibers for the production of a structured meat product. Further, one of ordinary skill in the art, armed with the teachings of this specification, would understand that other types of scaffolding may be used in the present invention. For example, Ben-Arye, et al. (Textured soy protein scaffolds enable the generation of three-dimensional bovine skeletal muscle tissue for cell-based meat. Nat Food 1 , 210-220 (2020). doi.org / 10.1038 / s43016-020-0046-5) teaches a soy protein-based scaffold for the production of cell-based meat. Likewise, International Patent Publication No. WO 2023 / 118872 A1 to Cellular Agriculture teaches a flexible polymer sheet having channels for the transport of cell culture medium.

[0075] It is contemplated that in the present invention the scaffolding takes up approximately 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the interior volume or of the lumen of the bioreactor.

[0076] Upon completed culture of a meat product inside the bioreactor of the present invention, the product can be harvested. This is accomplished by, in one example, 1 ) removing any plumbing from the bioreactor (or, removing the bioreactor from a culture control device); 2) snapping or causing to be snapped (or cutting or causing to be cut) one or both of the ends of the bioreactor off of the housing at the circumferential groove(s); and, 3) peeling or causing to be peeled the side walls of the housing by separating the sections of housing side wall from each other with a peeling motion (or equivalent) wherein the housing separates (at least partially) at theAttorney Docket No.: P24-154-SEC-WO01 longitudinal grooves into individual wall sections. After partial or complete removal of the housing from the cultured meat product, the housing and cultured meat product are separated from each other. If the housing is partially removed, the cultured meat product may be pushed out of the part remaining in the housing. In some scenarios, the ports may be used a leverage point to initiate the peeling of the housing wall ( / .e., a “peeling handle”). Peeling handles can also be, for example, tabs or loops built or molded into the outer wall of the reactor. They can also serve as a failure point indicator and as an aid to initiate peeling. Other means to initiate peeling may include applying a twisting of torsional force at the end or ends of the cell culture bioreactor of the present invention. In this case, an over torqued endcap may serve as type of “peeling handle.” The twisting or torsional force would cause the failure points at the grooves to begin to break partially separating the sections of the bioreactor housing from each other. This would be followed up with the “peeling” of the partially separated sections from each other.

[0077] In another example, the bioreactor lacks one or both of the circumferential grooves. In this design, the longitudinal grooves extend to the end of the housing at the first end, at the second end or both ends. With this design, the cultured meat product is harvested by 1) removing any plumbing from the bioreactor (or, removing the bioreactor from a culture control device); and 2) peeling or causing to be peeled the side walls of the housing by separating the sections of housing side wall from each other with a peeling motion (or equivalent) wherein the housing separates (at least partially) at the longitudinal grooves into individual wall sections. After partial or complete removal of the housing from the cultured meat product, the housing and cultured meat product are separated from each other. If the housing is partially removed, the cultured meat product may be pushed out of the partially remaining housing.

[0078] In one design, the ends of the bioreactor may be removable without snapping or cutting. That is, they be, for example, a friction fit, Luer® fit or screw fit over the ends of the housing and may be pulled off or screwed off of the housing at harvest time.Attorney Docket No.: P24-154-SEC-WO01

[0079] The bioreactor of the present invention, as discussed above, is made from one or more plastics. The bioreactor is molded from the plastic by methods well known to one of ordinary skill in the art of plastic manufacturing. The bioreactor can then be modified, if necessary or desired, by cutting, grinding, or the like, to the desired length and to modify or create the longitudinal and, if desired, circumferential grooves discussed, above.Exemplification

[0080] Certain embodiments of the present invention are given here to better describe the present invention but do not limit the scope of the invention in any way. One of skill in the art will be able to apply the teachings of this specification to related embodiments based on the teachings of this specification combined with the knowledge of an person of ordinary skill in the art. Numbers not repeated in subsequent figures are the same as previously indicated and left out merely for clarity.

[0081] Example 1

[0082] An example of the single-use cell culture bioreactor of the present invention 100 is shown in Fig. 1. As is seen, the device is an elongated, hollow structure ( / .e., having a lumen) with a first end 102 and a second end 104 and with a longitudinal dimension LD and a circumferential dimension CD. It also has two ports 106, 108 positioned at approximately 90 degrees from the length of the longitudinal direction of the bioreactor. The device may be made with more than two ports if needed or desired. The ports are in fluid communication with the lumen of the bioreactor. The first and second end of the reactor may also be open to the bioreactor lumen or may be sealed, depending of desired use. Also shown in Fig. 1 are groves in the outer wall of the bioreactor. Shown is one longitudinal groove 114. Another longitudinal groove is positioned on the opposite side of the bioreactor out of view. In this example of the bioreactor, the longitudinal grooves end at circumferential grooves 110, 112 proximal to the first and the second ends, respectively.

[0083] Fig. 2 shows a schematic diagram of cut-away views of a single-use cell culture bioreactor of the present invention. Fig. 2A shows an end view of the bioreactor housing of the present invention. Fig. 2B shows a longitudinal cross sectional view of a single-use cell culture bioreactor of the present invention. The lumen 116, wall 118 and the fluid connectivity of the twoAttorney Docket No.: P24-154-SEC-WO01 represented ports 106, 108 to the lumen is shown. Fig. 2C shows a cross sectional view highlighting the interior lumen 116 and wall 118 at the position of one of the ports. One circumferential groove 110 is shown. The groove allows for the end of the bioreactor to be “snapped” off of the bioreactor thereby allowing for easier harvesting of the cells and hollow fiber scaffolding in the lumen of the bioreactor. Fig. 2D shows a close-up view of an end of the single-use cell culture bioreactor of the present invention. Visible in this view are two longitudinal grooves 114A, 114B.

[0084] Figs. 3A and 3B show (A) a schematic representation of the single-use cell culture bioreactor of the present invention highlighting the circumferential 110, 112 and a longitudinal groove 114. The close-up image in the insert (B) is a cut-away view showing an intersection between a circumferential groove 110 and longitudinal grooves 114A, 114B. In this embodiment, the grooves leave a wall thickness in the wall of the elongated tubular element of the bioreactor of 0.4 mm.

[0085] Fig. 4 shows a single-use cell culture bioreactor of the present invention. In Fig. 4A, a bioreactor is shown prior to the ends of the bioreactor were snapped off. Fig. 4B shows the ends of the bioreactor snapper off. Fig. 5 shows two bioreactor housing wall sections as denoted by two longitudinal grooves in the housing (only one is visible) running between the two circumferential grooves, of the bioreactor. Fig. 5B shows the single-use cell culture bioreactor of the present invention where one of the bioreactor housing wall sections is partially separated from the other bioreactor housing wall section. The separation is along both of the longitudinal grooves starting at one of the circumferential grooves and ending nearly at the second circumferential groove. Fig. 5C shows the bioreactor after the first bioreactor housing wall section has been completely removed from the bioreactor.Although not shown here, the first bioreactor housing wall section can also be removed from the bioreactor after the first and / or the second end sections are snapped off at the circumferential grooves.

[0086] Fig. 6 is a micrograph showing plastic residue and plastic shards visible on the end of the hollow fiber bundle. These were caused by cutting the end of the bioreactor off and rendering any cultured meat productAttorney Docket No.: P24-154-SEC-WO01 potentially unfit for consumption. The present invention utilizing snap-off (or screw-off) endcaps solves this problem.

[0087] Fig. 7 shows four micrographs, two showing the fiber bundles and showing two cross-sectional views, of the harvested structured cultured meat product of the present invention with cell mass filling the voids between the hollow fibers.

[0088] Example 2

[0089] Here is provided an example of the use of the single-use cell culture bioreactor of the present invention. In this example the bioreactor of the present invention comprises hollow fibers as the structural element, i.e., a scaffolding matrix, for the growth of cells. However, one of skill in the art may wish to use other means of providing a scaffolding matrix. Numerous examples of cell culture scaffolding are provided in the art. See, above.

[0090] The single-use bioreactor of the present invention was sterilized, seeded with cells (for example, myocytes and adipocytes for the creation of a structured cultured meat product) and connected to a culture media source. The culture media provided nutrients and oxygen to the cells to enable cell divisional and cell growth. The culture media entered the bioreactor through an inlet port. The inlet port may be through the end of the housing or in the housing wall. The inlet port allowed for the media to be channeled into the interior of the hollow fibers, through the hollow fiber wall and into the interior of the bioreactor between the hollow fibers where the cells were seeded.Spent media with waste products from cellular metabolism exited through an exit port The exit port was located distal to the inlet port at the opposite end of the bio reactor housing. Larger bioreactors may require more than one inlet port and outlet port to ensure an even distribution of fresh media to the cells and an even removal of waste products. The cells are cultured until a confluency of at least 75% is reached. The confluency may reach higher levels, such as 80%, 85%, 90%, 95%, 98%, 99% and 100%, depending when the culture process is stopped. In this example, the confluency was over 80% when the culture was terminated.

[0091] Upon the termination of the cell culture process, the bioreactor was removed from both the feed and spent media lines. The cell culture mass (e.g., structured cultured meat product) was removed from the bioreactor byAttorney Docket No.: P24-154-SEC-WO01 removing the endcaps and / or snapping the ends of the bioreactor off at the circumferential grooves and then peeling a portion of the bioreactor wall from the cell culture mass. In one exemplification, the bioreactor had two longitudinal grooves separating the bioreactor housing into two sections. Upon peeling one of the sections off of the cell culture mass, the cell culture mass freely separated from the remaining wall of the bioreactor. The resultant structured cultured meat product is shown in Fig. 7.

[0092] Example 3

[0093] Here is provided exemplification of the force necessary to break or separate the sections of an embodiment of the bioreactor housing of the present invention. Fig. 9 shows three panels (A - C). The testing device is designed to apply a measurable force to the bioreactor housing of the present invention. The instrument is a Zwick / Roell 2.5kN RetroLine (Kennesaw, GA). This test was designed to duplicate the mechanism of force that would be used by an end user. Fig. 9A shows how a user would snap the end off of a bioreactor housing of the present invention how this motion will be simulated in the testing device. Fig. 9B shows a bioreactor housing of the present invention positioned in the testing device. Fig. 9C shows testing in process with a rate of travel by the vertical rod of 10 mm / min.

[0094] Fig. 10 shows a graph of the breaking force of a bioreactor housing of the present invention made of HIPS with several duplications. Approximately 15 pounds of force was necessary to break the ends off of the housing exemplified here indicating that the housing is rigid enough for use as a bioreactor, i.e., to be clamped and to withstand feed pressures, but also easily opened by a user.

Claims

Attorney Docket No.: P24-154-SEC-WO01We Claim:1 . A single use cell culture bioreactor housing, comprising: a. an elongated tubular element, said elongated tubular element having a first end and a second end, a wall defining a lumen and having an inside surface and an outside surface, said wall comprising at least two wall elements that are at least partially separable from each other along longitudinal separation lines running the length or substantially the length of the elongated tubular element; b. at least one fluid path through the bioreactor housing for carrying culture media into the bioreactor housing and spent media out of the bioreactor housing.

2. The bioreactor of Claim 1 , said elongated tubular element further comprises at least two longitudinal grooves at the longitudinal separation lines extending the length of the elongated tubular element or substantially the length of the elongated tubular element on the outside surface of said wall, said longitudinal grooves reducing the thickness of the wall by at least 50% as compared to the non-grooved wall of the housing.

3. The bioreactor of Claim 1 , said wall elements of said elongated tubular element are held together with snap-fit joints located at the longitudinal separation lines.

4. The cell culture bioreactor housing of Claims 1 - 3, further comprising a scaffolding matrix located in the lumen of the bioreactor housing.

5. The cell culture bioreactor housing of Claim 4, wherein said scaffolding matrix fills at least 30%, at least 40%, at least 50% or at least 60% of the lumen.

6. The cell culture bioreactor housing of any preceding claim, wherein said scaffolding matrix comprises a plurality of hollow fibers.Attorney Docket No.: P24-154-SEC-WO017. The cell culture bioreactor housing of any preceding claim, wherein said scaffolding is edible.

8. The cell culture bioreactor housing of Claim 6, wherein said hollow fibers extend from the first end to the second end of the elongated element of the housing.

9. The cell culture bioreactor housing of Claim 2, wherein said housing further comprises a circumferential groove on the outer surface of the wall proximal to the first and / or the second ends and intersecting with the longitudinal grooves.

10. The cell culture bioreactor housing of any preceding claim, further comprising removable endcaps attachable to and detachable from the first and / or second ends of the elongated tubular element.11 . The cell culture bioreactor housing of any preceding claim, wherein said elongated tubular element is selected from a cross-sectional shape of round, oval, ellipsoid, square and rectangular.

12. The cell culture bioreactor housing of any preceding claim, wherein the number of longitudinal lines are from two to ten.

13. The cell culture bioreactor housing of any preceding claim, wherein the width of the elongated tubular element is from 3 mm to 30 cm.

14. The cell culture bioreactor housing of any preceding claim, wherein the length of the elongated tubular element is from 6 cm to 3 meters.

15. The cell culture bioreactor housing of Claim 9, wherein said longitudinal and circumferential grooves leave about 0.2 mm to about 1 .0 mm of the wall thickness in the elongated tubular element.Attorney Docket No.: P24-154-SEC-WO0116. The cell culture bioreactor housing of Claim 9, wherein said longitudinal and circumferential grooves leave about 0.4 mm of the wall thickness in the elongated tubular element.

17. The cell culture bioreactor housing of Claim 9, wherein said longitudinal and circumferential grooves delineate bioreactor housing wall elements of the elongated tubular element and provide for a failure area or areas that allow for the at least partial separation of two or more of the wall elements from each other.

18. The cell culture bioreactor housing of Claim any preceding claim, wherein the wall elements are completely separatable from each other.

19. The cell culture bioreactor housing of any preceding claim, wherein said bioreactor housing is opaque.

20. The cell culture bioreactor housing of any preceding claim, wherein said bioreactor housing is transparent or translucent.

21. The cell culture bioreactor housing of any preceding claim, wherein said bioreactor housing is flexible.

22. The cell culture bioreactor housing of any preceding claim, wherein said bioreactor housing is biodegradable.

23. The cell culture bioreactor housing of any preceding claim, wherein said bioreactor housing is rigid or essentially rigid.

24. The cell culture bioreactor housing of any preceding claim, wherein said elongated tubular element comprises one, two or more ports in said wall to provide for said fluid path.Attorney Docket No.: P24-154-SEC-WO0125. The cell culture bioreactor housing of Claim 24, wherein, upon harvesting, said ports are used to provide leverage for initiating the separation of the bioreactor housing wall elements.

26. The cell culture bioreactor housing of any preceding claim, wherein said elongated tubular element is molded from one or more plastics.

27. The cell culture bioreactor housing of any preceding claim, wherein said elongated tubular element is made of two or more bonded sections.

28. The cell culture bioreactor housing of any preceding claim, wherein said bioreactor is a single-use bioreactor.

29. The cell culture bioreactor of Claim 9, wherein said longitudinal and circumferential grooves are molded, extruded, cut or both.

30. A single use cell culture bioreactor housing, comprising: a. an elongated tubular element, said elongated tubular element having at least a first end and a second end, a wall having an inside surface and an outside surface, said wall defining a lumen, and said wall comprising at least two wall elements; b. at least one fluid path through the bioreactor housing for carrying culture media into the bioreactor housing and spent media out of the bioreactor housing; c. wherein said wall elements are reversibly attachable to each other and are held together with snap-fit joints.

31. A single use cell culture bioreactor housing, comprising: a. an elongated tubular element, said elongated tubular element having a first end and a second end, a wall having an inside surface and an outside surface, said wall defining a lumen; b. at least one fluid path through the bioreactor housing for carrying culture media into the bioreactor housing and spent media out of the bioreactor housing;Attorney Docket No.: P24-154-SEC-WO01 c. wherein said bioreactor housing further comprises at least two longitudinal grooves extending the length of the elongated tubular element or substantially the length of the elongated tubular element on the outside surface of said wall, said longitudinal grooves reducing the thickness of the wall by at least 50% as compared to the non-grooved wall of the housing.

32. A method of culturing and harvesting a cell culture product, the method comprising: a. providing; a single use bioreactor housing of any of the preceding claims; b. seeding interior space of the single use bioreactor with one or more cell types; c. culturing the cells by providing culture media to the cells and removing spent media from the cells via the fluid path until the cell culture reaches about 75% confluency or greater to create a structured cell culture product; d. stopping the flow of culture media into and the flow of spent media out of the bioreactor; e. separating, at least partially, one wall element elongated tubular element from another wall element from the structured cell culture product thereby permitting the removal of the structured cell cultured product from the bioreactor housing.

33. A method of culturing and harvesting a cell culture product, the method comprising: a. providing; a single use cell culture bioreactor having an elongated tubular element, said elongated element having 1) a first end and a second end, a wall having an inside surface and an outside surface and a lumen; 2) at least one fluid path through the bioreactor housing for carrying culture media into the bioreactor and spent media out of the bioreactor housing; 3) wherein said bioreactor housing further comprises at least two wall elements that are at least partially separable from each other along two or more longitudinal separationAttorney Docket No.: P24-154-SEC-WO01 lines between said first and said second ends, and; 4) a scaffolding material in the interior space of the elongated tubular element; b. seeding interior space of the single use bioreactor with one or more cell types; c. culturing the cells by providing culture media to the cells and removing spent media from the cells via the fluid path until the cell culture reaches about 75% confluency or greater to create a structured cell culture product; d. stopping the flow of culture media into and the flow of spent media out of the bioreactor; e. separating, at least partially, one wall element elongated tubular element from another wall element from the structured cell culture product thereby permitting the removal of the structured cell cultured product from the bioreactor housing.

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