Method for culturing muscle cells using a perfusion bioreactor

Abstract

Provided herein are methods for culturing muscle cells for use in edible products, including seeding muscle cells onto one or more porous hollow fibers; maintaining a perfusion module with the one or more porous hollow fibers oriented at an angle of 0 to 90 degrees from horizontal for a period of time sufficient for the muscle cells to initially attach to the exterior surface of each of the one or more porous hollow fibers; rotating the perfusion module; connecting the perfusion module to a perfusion bioreactor system; and culturing the muscle cells attached to the exterior surface of the porous hollow fibers. Also included are edible products produced by such methods.

Description

[Technical Field]

[0001] The present invention provides methods for culturing muscle cells, particularly using a hollow fiber bioreactor (HFB). Also provided are edible products comprising or formed from the cells produced by the method or the cells and hollow fibers used in the method. [Background technology]

[0002] There is growing interest in cultured meat as a protein alternative. While laboratory-scale production of cultured meat in its simplest form—muscle cells or co-culture of muscle cells with fat cells—has been achieved, scaling up the process to create a viable, economical product remains challenging.

[0003] Challenges to be overcome include ethical sourcing of raw materials, reducing the cost of cell culture media, increasing protein yield in muscle cell culture, improving energy efficiency and resource use in the bioprocess itself, and valorizing waste.

[0004] For improved process efficiency and reduced environmental impact, higher cell density bioreactors allow for smaller culture volumes, reducing space requirements, labor required for cell placement and harvesting, and the amount of raw materials required for production. Smaller bioreactors also require less power and utilities, resulting in lower operating costs.

[0005] The structure of native skeletal muscle consists of multiple arrays of uniaxial striated muscle fibers interlocked with adipocytes, fibroblasts, capillaries, and veins. Capillaries connect most of the muscle fibers within the muscle bundles, providing blood perfusion, providing sufficient oxygen and nutrients to muscle cells, and removing cellular metabolic waste products. This structure is well reproduced in hollow fiber bioreactors (HFBs), where an inlet medium carries oxygen and nutrients through the fibers to nourish the cells, and a perfusion flow (permeate) and / or retentate carries waste products away at the outlet.

[0006] Although the HFB concept appears to meet the target milestones of higher cell density and vascularized architecture in whole-cut meat, practical challenges remain to be overcome, including:

[0007] Hollow fiber bioreactors are classified as hydraulic bioreactors, meaning that mixing is achieved by fluid flow rather than mechanical mixing. In this method, cells are seeded into a matrix containing porous hollow fibers, allowing the cells to adhere to the surface of the fibers and circulate the medium. Hollow fiber systems have the advantages of low shear stress, high selectivity for transported nutrients, and being ideal for metabolically intensive cell types. However, hollow fiber bioreactors are primarily limited to cell culture and are not compatible with the cultivation of tissue scaffolds.

[0008] Baba K, Sankai Y., Development of a biomimetic system for scaling up cell spheroids - a component of cell transplantation, 2017 39th Annu Int Conf IEEE Eng Med Biol Soc.2017.10.1109 / EMBC.2017.8037147; Yamamoto Y, Ito A, Jitsunobu H, Yamaguchi K, Kawabe Y, Mizumoto H, Kamihira M., Hollow fiber bioreactor perfusion culture system for magnetic force-based skeletal muscle tissue engineering, J Chem Eng Japan.2012;45:348-54.https: / / doi.org / 10.1252 / jcej.11we237; and Bettahalli NMS, Vicente J, Moroni L, Higuera GA, Van Blitterswijk CA, Wessling M, Stamatialis DF, Integration of hollow fiber membranes improves nutrient delivery in three-dimensional tissue structures, Acta Biomater [Internet], Acta Materialia Inc.;2011;7:3312-24 all in a perfusion bioreactor C2C 12The use of hollow fibers for culturing cells has been disclosed. However, all of these disclose that the cells are not attached to the hollow fibers, but are attached or maintained on a separate scaffold or matrix. The hollow fibers are used only to supply medium to the cells, and are not used for support or scaffold purposes.

[0009] Bettahalli NMS, Steg H, Wessling M, Stamatialis D, Development of Poly(L-lactic acid) Hollow Fiber Membranes for Artificial Vascular Tissue Engineering Scaffolds, J Memb Sci [Internet], Elsevier BV;2011;371:117-26, discloses the use of a PLLA hollow fiber system for culturing C2C12 cells. The fibers are used to supply medium to the cells and as a scaffold for cell attachment. However, continuous medium supply resulted in a decrease in cell number (i.e., the cells were unable to remain attached and grow in the bioreactor).

[0010] Luetchford, KA; Wung, N.; Argyle, IS; Storm SP; Weston SD; David Tosh D.; Ellis MJ (50%), Next-generation in vitro liver model design: coupling permeable polystyrene membranes with transdifferentiated cell lines, Journal of Membrane Science. 2018, 565, pp. 425-438, discloses the use of hollow fibers for hepatocyte culture for in vitro models. However, there is no suggestion of using such a system for scaled cell growth or myocytes, or how to adapt these methods to myocytes.

[0011] There is a need for improved systems and methods for culturing muscle.

[0012] There is a need for improved methods of culturing muscle cells to produce meat analogs.

[0013] There is also a need for improved methods for continuous culture of muscle cells or for increasing cell yield. (Brief summary of disclosure)

[0014] The present invention is based on the surprising discovery that the methods described herein can be used to produce high yields of muscle cells suitable for use in the production of meat analogs. For example, it has been found that maintaining the prefusion module at an angle of about 0-90 degrees for a predetermined period of time causes cells to attach to the hollow fibers. It has also been found that culturing cells at an angle of about 0-90 degrees results in greater cell production.

[0015] In one aspect of the invention, there is provided a method of culturing muscle cells for an edible product, the method comprising: a) providing a perfusion module for a perfusion bioreactor system; the perfusion module comprising one or more porous hollow fibers having an outer surface and an internal lumen (lumen); b) seeding muscle cells onto the one or more porous hollow fibers; c) maintaining the perfusion module with the one or more porous hollow fibers oriented at an angle of 0 to 90 degrees from horizontal for a period of time sufficient for the muscle cells to initially attach to the outer surface of each of the one or more porous hollow fibers; d) rotating the perfusion module; e) connecting the perfusion module to a perfusion bioreactor system; and f) culturing the muscle cells attached to the outer surface of each of the one or more porous hollow fibers under conditions suitable for proliferation and / or differentiation of the muscle cells.

[0016] In certain embodiments, the period is at least 10 minutes.

[0017] In certain embodiments, the angle is 0 to 30 degrees from horizontal. In certain embodiments, the angle is 5 to 20 degrees from horizontal. In certain embodiments, the angle is 8 to 10 degrees from horizontal. In some embodiments, the angle is approximately 9.2 degrees.

[0018] In certain embodiments, rotating comprises applying a centrifugal force of 0 to 50 N. For example, centrifugal force is applied to the perfusion module and cells seeded therein.

[0019] In certain embodiments, the rotating comprises continuously rotating. In certain embodiments, the rotating comprises continuously rotating at a speed of 0-30 rpm. In certain embodiments, the rotating comprises continuously rotating at a speed of about 1-4 rpm. In certain embodiments, the rotating comprises continuously rotating for at least 1 hour. In certain embodiments, the rotating comprises continuously rotating for at least 3 hours. In certain embodiments, the rotating comprises continuously rotating for about 3-5 hours.

[0020] In certain embodiments, rotating the perfusion module includes rotating the perfusion module with an offset between a midpoint of a centerline of the perfusion module and an axis of rotation. In certain embodiments, the offset is greater than 0 mm. In certain embodiments, the offset is at least 10 mm. In certain embodiments, the offset is up to 500 mm. In certain embodiments, the offset is about 150 mm.

[0021] In certain embodiments, the attachment efficiency is at least 5%, as determined quantitatively by direct or indirect measurement of viable cells attached to the hollow fiber. In certain embodiments, the attachment efficiency is at least 10%, 20%, 30%, 40%, or 50%, as determined quantitatively by direct or indirect measurement of viable cells attached to the hollow fiber. In certain embodiments, the attachment efficiency is at least 55%, as determined quantitatively by direct or indirect measurement of viable cells attached to the hollow fiber.

[0022] In certain embodiments, rotating the perfusion module includes intermittently rotating the perfusion module, ie, at a speed of 0 to 30 rpm for about 1 second to about 5 minutes every about 10 seconds to about 30 minutes for a total time of at least 1 hour.

[0023] In certain embodiments, rotating the perfusion module includes rotating at a speed of about 12 rpm for about 30 seconds about every 5 minutes.

[0024] In certain embodiments, rotating the perfusion module comprises rotating for at least 3 hours.

[0025] In certain embodiments, prior to seeding, the muscle cells (i) washing with a composition comprising a cell exfoliant; (ii) harvested in a culture medium containing a growth promoter; (iii) centrifuging to form a pellet; and (iv) Resuspending in culture medium to form a cell suspension.

[0026] In certain embodiments, seeding the muscle cells comprises applying a cell suspension comprising the muscle cells to the perfusion module.

[0027] In certain embodiments, seeding the muscle cells comprises applying a cell density at least about 0.1 times higher than that used on 2D tissue culture plastic, e.g., 0.1 to 10 times higher.

[0028] In certain embodiments, seeding the muscle cells is at least 1000 cells / cm 2 applying the cells to the perfusion module.

[0029] In certain embodiments, the one or more porous hollow fibers comprise: It is hydrophilic; be washable; and / or It is reusable.

[0030] In certain embodiments, the one or more porous hollow fibers comprise polystyrene.

[0031] In certain embodiments, the one or more porous hollow fibers are biodegradable and / or edible.

[0032] In certain embodiments, culturing includes pumping a culture medium through the perfusion bioreactor system and the interior lumen of each of the one or more porous hollow fibers; optionally, culturing further includes maintaining the perfusion module in an orientation in which the one or more porous hollow fibers are at an angle of 0 to 90 degrees from horizontal. Optionally, the angle is 0 to 30 degrees; preferably 5 to 20 degrees; more preferably 8 to 10 degrees from horizontal.

[0033] In certain embodiments, culturing comprises pumping culture medium through the perfusion bioreactor system and the interior lumen of each of the one or more porous hollow fibers at a rate of at least 10 μL / hr / hollow fiber.

[0034] In certain embodiments, culturing comprises maintaining the muscle cells at a temperature of at least 15° C. and / or 5% CO 2 .

[0035] In certain embodiments, culturing comprises pumping culture medium through said perfusion bioreactor system and perfusion module for at least 3 hours.

[0036] In certain embodiments, culturing comprises pumping a first culture medium through the perfusion bioreactor system and the interior lumen of each of the one or more porous hollow fibers, optionally wherein the first culture medium is a growth medium.

[0037] In certain embodiments, culturing comprises pumping a second culture medium through the perfusion bioreactor system and the interior lumen of each of the one or more porous hollow fibers, optionally wherein the second culture medium is a differentiation medium; further optionally, the differentiation medium is perfused through the bioreactor system and perfusion module for at least 3 hours.

[0038] In certain embodiments, the perfusion bioreactor system comprises devices for monitoring metabolite and / or oxygen concentrations in the culture medium.

[0039] In certain embodiments, the method further comprises: (g) harvesting the muscle cells from the perfusion module: Includes:

[0040] In certain embodiments, the harvested muscle cells are formed into a cultured meat product; or, the one or more hollow fibers are edible, and the harvested cells and the one or more porous hollow fibers are formed into a cultured meat product.

[0041] In certain embodiments, the muscle cells are derived from at least one food animal cell; optionally, the muscle cells comprise one or more of fibroblasts, skeletal muscle cells, smooth muscle cells, and / or myoblasts; further optionally, the muscle cells are derived from one or more of non-human embryonic stem cells and / or pluripotent stem cells.

[0042] In another aspect of the present invention, there is provided an edible product comprising muscle cells obtained by the methods described herein.

[0043] In another aspect of the present invention, there is provided an edible product obtainable by the methods described herein.

[0044] In certain embodiments, the edible product is a cultured meat product.

[0045] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including but not limited to" and are not intended to exclude, and do not exclude, other parts, additives, components, elements or steps.

[0046] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, it is understood that the specification contemplates the plural as well as the singular, unless the context otherwise requires.

[0047] It is to be understood that any feature, element, property, compound, chemical moiety or group described in connection with a particular aspect, embodiment or example of the invention is also applicable to the other aspects, embodiments or examples described herein, unless there is a contradiction thereto.

[0048] Various aspects of the invention are described in further detail below. [Brief explanation of the drawings]

[0049] Hereinafter, embodiments of the present invention will be further described with reference to the accompanying drawings. Figure 1 shows the in vivo-like environment of the HFB. Cells are seeded on the outside of the porous fibers. Culture medium is delivered through the interior lumen of the fibers, which mimics capillaries. (A) Longitudinal cross section of the fiber (not to scale). (B) Cross section of a three-fiber reactor.

[0050] [Figure 2] Figure 2 shows the HFB module. (A) Module dimensions. The dimensions are selected to fit three fibers. Different sizes can be manufactured and adjusted to fit individual systems and fibers. (B) Photograph of the module with end caps and module connectors attached.

[0051] [Figure 3] Figure 3 shows the fabrication of the module. (A) The fibers are cut to size and inserted into the module. (B) The fibers are glued to the module and allowed to dry. (C) The ends are cut flush with the glass.

[0052] [Figure 4] Figure 4 shows the setup of the HFB system. The arrows indicate the direction of media flow. A = supply tube. B = pump tube. E = HFB module. D = holding tube with clamp. C = permeate tube.

[0053] Figure 5 shows an indirect measure of metabolic activity of RSkMC on tissue culture plastic (TCP) and hydrophilic polystyrene (PX40) flat-sheet membranes. Metabolic activity was assessed using the resazurin assay. Data represent the mean ± SD, n = 1, N = 6.

[0054] FIG. 6 shows the relationship between the midpoint of the (axial) centerline of the perfusion module and the axis of rotation when determining the offset.

[0055] The patent, scientific and technical literature referred to herein establishes knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of issued patents, published and pending patent applications, and other publications cited herein are incorporated by reference to the same extent as if each were specifically and individually indicated to be incorporated by reference. In the case of conflict, the present disclosure will control.

[0056] Various aspects of the invention are described in further detail below. (Detailed explanation)

[0057] The methods described herein involve culturing the cells described herein using a hollow fiber perfusion bioreactor.

[0058] Perfusion culture refers to a continuous culture method in which cells are maintained within or pumped back into a bioreactor. The cell culture medium that is perfused through the bioreactor does not contain the cells. Perfusion-based culture methods, such as those considered for continuous stirred-tank reactors, can reduce the working volume while increasing cell concentration and production within the reactor.

[0059] As used herein, the term "perfusion bioreactor" refers to a cell culture system in which cell culture medium (e.g., first cell culture medium, second cell culture medium, culture medium, cell proliferation cell culture medium, and / or cell differentiation cell culture medium) is continuously exchanged with fresh medium. A perfusion bioreactor system can include means (e.g., an outlet, an inlet, a pump, or other such device) for periodically or continuously withdrawing and adding substantially the same amount of replacement cell culture medium to the bioreactor. The addition of replacement liquid culture medium can be performed substantially simultaneously with or immediately after the removal of the initial cell culture medium from the bioreactor. The means for removing liquid culture medium from the bioreactor and the means for adding replacement liquid culture medium can be a single device or system. For example, the means for removing and exchanging (i.e., perfusing the cell culture medium) can be a peristaltic pump system.

[0060] A "bioreactor" can be any device or system that maintains a biologically active environment, such as a chamber or vessel in which cells can be cultured. There are several different types of bioreactors that vary in shape (e.g., cylindrical), size (milliliters, liters, to cubic meters), and material (e.g., stainless steel, glass, plastic). Bioreactors are therefore suitable for growing cells or tissues in cell culture. Bioreactors can be configured to accept hollow fibers as described herein. A hollow fiber perfusion bioreactor (HFB) includes a perfusion module (also called a bioreactor) containing hollow fibers.

[0061] Typical HFBs and perfusion systems for use in the perfusion modules described herein include those described, for example, in Luetchford, KA; Wung, N.; Argyle, IS; Storm SP; Weston SD; David Tosh D.; Ellis MJ (50%), Next generation in vitro liver model design: Combining a permeable polystyrene membrane with a transdifferentiated cell line. Journal of Membrane Science. 2018. 565. pp. 425-438, and Wung, N., Acott, SM, Tosh, D., Ellis, MJ, Hollow fiber membrane bioreactors for tissue engineering applications. Biotechnol Lett. 36(12) 2357-2366 (2014).

[0062] Typically, hollow fiber perfusion bioreactor systems used in the methods described herein comprise a continuous perfusion system with constant nutrient replenishment and waste removal. Culture medium circulates through a perfusion module containing porous hollow fibers. The interior of the hollow fibers is sometimes referred to as the intracapillary space (ICS) or lumen, and the exterior is sometimes referred to as the extracapillary space (ECS). Cells grow within the ECS and attach and adhere to the exterior (external surface) of the fibers. Fibers can provide a large surface area for cell contact in a relatively compact system. A schematic diagram of the general concept of a hollow fiber system is shown in Figure 1. As shown in Figure 1, culture medium (1) flows through the internal lumen (10) of hollow fibers (12). Pores (14) in the hollow fibers allow nutrients to be transferred to cells (16) attached and adhered to the outer surface (18) of the hollow fibers.

[0063] Typically, low-molecular-weight cellular nutrients and metabolic waste products can pass between the ICS and ECS through the pores of the porous hollow fibers, while cells can be trapped within the ECS. One or more "side" ports can be used to introduce high-molecular-weight nutrients and / or flowing media or liquids into the ECS and provide a predetermined shear rate to the cells. Additional side ports can be used to collect fluids flowing through the ECS. The HFB provides a near-in vivo environment by allowing the fibers to mimic capillaries, protecting cells from the shear stresses associated with dynamic media delivery while also allowing predetermined shear to be applied to cells via fluid flow through the side ports as needed. This creates a versatile culture system with excellent mass transport, enabling high cell densities to be achieved.

[0064] Examples of perfusion modules are shown in Figures 2 and 3. Figure 2A shows a schematic diagram of an example perfusion module. The perfusion module (2) includes an inlet port (20) and an outlet port (21) located at opposite ends of a channel (22). The example shown also includes a first side port (23) and a second outlet port (24). Figure 3 shows a perfusion module with a hollow fiber (3) inserted into the perfusion module channel (32).

[0065] An example of a perfusion system setup is shown in Figure 4. The system includes a pump system (4) connected to a perfusion module (40 and E). Culture medium is supplied from a reservoir (42) via a feed tube (A) to the pump system (4), which then pumps the medium to the perfusion module via a pump tube (B). The culture medium passes through the perfusion module and exits the inner lumen of the hollow fiber via the retentate tube (D). The exchanged medium passes through the pores of the hollow fiber and enters the ECS via the permeate tube (C).

[0066] The pump system may be any suitable pump. For example, the pump system may be a peristaltic pump system or a vacuum pump system. The flow rate of the culture medium flowing through the perfusion module may be controlled by the pump system. The flow rate may be selected depending on the number of hollow fibers used, the cells being cultured, and / or the number of cells seeded on the outer surface of the hollow fibers.

[0067] For example, the culture medium can be pumped at a rate of at least 1 μL / hour per hollow fiber. That is, for each hollow fiber used, the flow rate is 1 μL / hour. In some examples, the flow rate is at least 10 μL / hour per hollow fiber. For example, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400 μL / hour per hollow fiber. In some examples, the flow rate is 100-300 μL / hour per hollow fiber. In some examples, the flow rate is 240-280 μL / hour per hollow fiber. In some examples, the flow rate is approximately 267 μL / hour per hollow fiber.

[0068] The perfusion bioreactor system may further comprise a monitoring device, which refers to a device suitable for determining and / or monitoring changes in one or more properties of the cells being cultured and / or changes in one or more properties of the perfusion medium after passing through the hollow fibers (retentate) and / or the perfusion medium after passing through the pores (permeate).

[0069] For example, the monitoring device may be a system for monitoring the nutrient and / or metabolite content of the permeate and / or retentate. As used herein, permeate refers to the culture medium that has passed through the pores of the hollow fiber (i.e., permeated the hollow fiber). As used herein, retentate refers to the culture medium that flows through the inner lumen of the hollow fiber (i.e., is retained within the hollow fiber). The nutrients that can be monitored may be any nutrients and / or metabolites contained in the culture medium used. For example, the nutrients and / or metabolites may be one or more of sugars (e.g., glucose), lactic acid (lactate), glutamine, glutamate, ammonia (ammonium ion), essential and non-essential amino acids, growth factors, albumin, attachment proteins, vitamins, oxygen, and / or carbon dioxide, etc.

[0070] The concentrations of nutrients and / or metabolites can be compared to the initial concentrations of the corresponding nutrients and / or metabolites in the culture medium before the cells were cultured or before it was perfused through the hollow fibers. Changes in the concentrations of nutrients and / or metabolites in the permeate and / or retentate compared to the initial cell culture medium can provide information regarding the growth rate, cell number, and / or state (e.g., viability and / or health) of the cells being cultured.

[0071] In some cases, the system for monitoring nutrient and / or metabolite concentrations may be separate from the perfusion bioreactor system. For example, the method may further include collecting a portion of the permeate and / or retentate at predetermined time points and determining the nutrient and / or metabolite concentrations. In some cases, the perfusion bioreactor system may include a nutrient and / or metabolite monitoring system directly connected to the perfusion bioreactor system.

[0072] Suitable methods for determining the concentrations of nutrients and / or metabolites in culture media are well known in the art. Kits, such as those provided by Megazyme, are available and include instructions. For example, for nutrients and / or metabolites such as sugars (e.g., glucose), lactic acid (lactate), glutamine, glutamate, and ammonia (ammonium ion), HPLC and / or GC-MS can be used to determine the concentrations of essential and non-essential amino acids, growth factors, albumin, and adhesion proteins. For example, oxygen and carbon dioxide monitors include those available from PreSens.

[0073] In some examples, the monitoring device can include a system for monitoring the dissolved oxygen concentration in the permeate and / or retentate. The dissolved oxygen concentration can be compared to the initial concentration of dissolved oxygen in the culture medium before the cells are cultured or before it is perfused through the hollow fibers. Changes in the dissolved oxygen concentration in the permeate and / or retentate compared to the initial cell culture medium can provide information about the growth rate, cell number, and / or status (e.g., viability and / or health) of the cells being cultured.

[0074] Systems for measuring dissolved oxygen concentration in culture media are well known, including the PreSens flow-through cell and the PreSens Fibox4 oxygen reader.

[0075] The cells seeded onto the hollow fibers include muscle cells. Muscle cells include cells that make up the contractile tissue of animals or cells that can differentiate into muscle cells. Muscle cells are derived from the mesodermal layer of embryonic germ cells. Mature muscle cells contain contractile filaments that move past each other, changing the size of the cell. They are classified as skeletal, cardiac, or smooth muscle. As used herein, the term "cells capable of differentiating into muscle cells" refers to stem cells and muscle progenitor cells that can differentiate into muscle cells (such as mature muscle cells).

[0076] Muscle cells include cells normally present in muscle tissue, including smooth muscle cells, cardiac muscle cells, skeletal muscle cells (e.g., muscle fibers or myocytes, myoblasts, myotubes, etc.), and any combination thereof. Muscle cells can include myoblasts, myotubes, myofibrils, and / or satellite cells.

[0077] The cells may further include adipocytes or adipocytes, which include any cell or group of cells contained in adipose tissue, including, for example, adipocytes, adipocyte precursor cells, preadipocytes, and mesenchymal stem cells.

[0078] The cells may be derived from any animal. Because the perfusion modules described herein may be used to produce edible products, the cells may not be derived from humans. In some examples, the cells may be derived from cattle, sheep, horses, pigs, goats, birds, fish, insects, crustaceans, cephalopods, mollusks, and / or camelids. Preferably, the cells may be derived from cattle, pigs, birds, and / or sheep. For example, the cells may be derived from cattle, pigs, chickens, fish, squid, insects, oysters, and / or sheep.

[0079] The cell culture medium that can be used in the methods described herein can be any suitable cell culture medium. The cell culture medium can be selected depending on the type of cells being cultured. Examples of culture media that can be used include Minimum Essential Medium (MEM, Sigma, St. Louis, MO), Dulbecco's Modified Eagle's Medium (DMEM, Sigma), Ham's F10 medium (Sigma), cell culture medium (Hyclone, Logan, UT), RPMI-1640 culture medium (Sigma), and chemically defined (CD) culture medium (formulated for individual cell types), such as CD-CHO culture medium (Invitrogen, Carlsbad, CA). The above culture solutions can be supplemented with supplementary components or ingredients as needed, including any components at the appropriate concentrations or amounts required or desired.

[0080] The above culture medium can be supplemented with auxiliary components or ingredients as needed, and the culture medium can contain one or more additives such as antibiotics, proteins, amino acids, sugars, etc.

[0081] "Culture medium" and "cell culture medium" refer to a nutrient source used to grow or maintain cells. As will be understood by those skilled in the art, a nutrient source may include components necessary for cells to grow and / or survive, or may include components that aid in the growth and / or survival of cells. Vitamins, essential or non-essential amino acids, trace elements, and surfactants (e.g., poloxamer) are examples of medium components. The media provided herein may also be supplemented with any one or more of insulin, plant hydrolysates, and animal hydrolysates.

[0082] "Culturing" cells refers to contacting the cells with cell culture medium under conditions suitable for the survival and / or growth and / or proliferation of the cells.

[0083] Perfusing the cell culture medium can include perfusing a first culture medium followed by one or more second cell culture media. The first cell culture medium is a cell culture medium for growing cells and may also be referred to as a growth medium.

[0084] Growth media can be media containing nutrient sources such as vitamins, minerals, carbon sources, energy sources, and other beneficial compounds that promote biochemical and physiological processes occurring during cell expansion or growth. Growth media can include one or more carbon sources, vitamins, amino acids, and inorganic nutrients. Exemplary carbon sources include monosaccharides, disaccharides, and / or starch. For example, growth media can include one or more carbohydrates, such as sucrose, fructose, maltose, galactose, mannose, and lactose. Growth media can also include amino acids. Suitable amino acids include those commonly incorporated into proteins as well as amino acids not commonly incorporated into proteins, such as argininosuccinic acid, citrulline, canavanine, ornithine, and D-stereoisomers. Growth media can also include growth promoters, such as serum, e.g., fetal bovine serum (FBS). Other examples of growth promoters include growth factors (e.g., recombinant growth factors), bovine eye fluid, sericin protein, and heat-inactivated coelomic fluid from earthworms. In some cases, the growth medium is a serum-free culture medium, and can optionally contain additional components depending on the cells being cultured. For example, serum-free culture media include those commercially available from ThermoFisher, Lonza Bioscience and Merck. The growth medium can also contain antibiotics.

[0085] For example, the growth medium may be Dulbecco's Modified Eagle's Medium (DMEM), which may contain 10% (V / V) filter-sterilized fetal bovine serum and 1% (V / V) penicillin / streptomycin solution.

[0086] In some cases, for example, when the cells are of insect origin, the growth medium may be a medium such as Gibco insect medium available from ThermoFisher.

[0087] The cells may be maintained and cultured in the growth medium for at least 3 hours, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 125, 130, 135, 140, 145, or 150 hours. In some cases, the cells may be continuously maintained in growth medium.

[0088] The cell culture medium may be changed to a second cell culture medium. The second cell culture medium may be a differentiation medium. A differentiation medium refers to a medium designed to support cell differentiation, i.e., the process by which cells change from one cell type to another. The differentiation medium may include one or more amino acids, antibiotics, vitamins, salts, minerals, or lipids. The differentiation medium may include at least one carbon source, such as a sugar. For example, glucose. The differentiation medium may also include one or more proteins, amino acids, or other additional acids. In some cases, the differentiation medium includes one or more growth promoters, such as serum, e.g., fetal bovine serum or horse serum. Other examples of growth promoters include growth factors (e.g., recombinant growth factors), bovine eye fluid, sericin protein, and heat-inactivated earthworm coelomic fluid. In some cases, the differentiation medium may be serum-free (serum-free culture medium) and may optionally include additional components depending on the cells being cultured. For example, serum-free media include those commercially available from ThermoFisher, Lonza Bioscience, and Merck. In some instances, the differentiation medium may be high glucose DMEM (97%) supplemented with 2% horse serum and 1% penicillin / streptomycin solution.

[0089] The cells may be maintained and cultured in the differentiation medium for at least 3 hours, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 125, 130, 135, 140, 145, or 150 hours.

[0090] In some cases, the culture medium may be changed multiple times. For example, cells may first be cultured in a growth medium, and then the culture medium may be switched to a differentiation medium. The differentiation medium may then be switched to a growth medium. The growth medium may be the same as before, or may contain different components and / or concentrations of components than the initial growth medium.

[0091] In some cases, cells may be continuously cultured. That is, the cells are maintained in one or more culture media as described herein. The cells may be cultured in a first medium for a first period (e.g., at least 3 hours or more), then in a second medium for a second period (e.g., at least 3 hours or more), and then in an additional medium, continuously culturing with any number of changes of culture medium, allowing the cells to constantly proliferate and / or differentiate. In such cases, the cells may be harvested at a predetermined cell density or time point to avoid overcrowding, loss of viability, and / or cell death.

[0092] Cell culture conditions (e.g., either proliferation or differentiation, or both) can be selected depending on the type and / or source of the cells. In some cases, cells can be cultured (e.g., proliferation and / or differentiation) at a temperature of at least 15°C. In some cases, cells can be cultured at a temperature of at least 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.

[0093] For example, when culturing insect cells, the cells can be cultured at a temperature between 15°C and 32°C.

[0094] For example, when culturing mammalian cells, the cells may be cultured at a temperature of 37°C.

[0095] For example, in the case of marine animal cells (such as crustacean, fish, or mollusk cells), the cells may be cultured at a temperature between 15° C. and 32° C. In some cases, marine animal cells may be cultured at a temperature between 15° C. and 30° C.

[0096] The cells may be cultured under defined atmospheric conditions. For example, the cells may be cultured in an atmosphere having a predetermined humidity and / or gas concentration. For example, the cells may be cultured in an atmosphere containing at least 5% CO2.

[0097] The polymer that can be used to form the hollow fibers can be any polymer suitable for culturing and / or maintaining cells. Suitable polymers include biodegradable polymers. The polymer can be a biocompatible polymer. A biodegradable polymer is any polymer that can be degraded by a biological system, such as a polymer that can be broken down into harmless products by the action of living organisms. A biocompatible polymer, along with its metabolites or degradation products, is generally non-toxic to cells or recipients (e.g., humans or animals) and does not have a significant adverse effect on the cells or recipients at concentrations resulting from the polymer's degradation. Generally speaking, a biocompatible polymer is one that does not adversely affect the health of cells or recipients. Because one application of the hollow fibers described herein is the production of edible products, biocompatible and / or biodegradable polymers can be advantageous when the fibers or portions thereof are intended for human consumption (e.g., ingested).

[0098] Biodegradable polymers include linear aliphatic polyesters such as polylactic acid, polyglycolic acid, polycaprolactone, polyhydroxybutyric acid, polyhydroxyvalerate, and their copolymers within the aliphatic polyester family such as poly(lactic acid-co-glycolic acid) and poly(glycolic acid-co-caprolactone), copolymers of linear aliphatic polyesters with other polymers such as poly(glycolic acid-co-trimethylene carbonate) copolymers, poly(lactic acid-co-lysine) copolymers, tyrosine-based polyarylates or polyiminocarbonates. Poly(ester-ether)s such as poly(ester-amide)s, ...

[0099] The polymer may be an edible polymer, which refers to any polymer that is acceptable for use in edible products.

[0100] Examples of edible polymers include synthetic polymer compounds such as polyvinyl alcohol, carboxyvinyl polymers, hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose, ethylcellulose, low-substituted hydroxypropylcellulose, crystalline cellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, and sodium carboxymethylstarch, as well as polymer compounds obtained from natural products such as sodium alginate, dextran, casein, pullulan, pectin, guar gum, xanthan gum, tragacanth gum, acacia gum, zein, gelatin, chitin and chitosan, silk, fibrin, starch, or soybeans.

[0101] The use of edible polymers may provide hollow fibers and products containing hollow fibers that are edible, for example, cell cultures grown on hollow fibers may provide edible products that do not require removal of the hollow fibers prior to consumption.

[0102] The hollow fibers may be made of a digestible polymer. "Digestable" refers to a material that, when ingested by a subject, can be broken down into compounds that can be absorbed and used as nutrients or excreted from the subject's body. Digestible polymers include BCS, polylactic acid (PLA), synthetic polyamides, polycarbonates, polyisocyanurates (PIR), polyurethanes, polyethers, proteins, polysaccharides (such as starch), polylactones, polylactams, glycols, and the like.

[0103] In some instances, the hollow fibers include poly(lactic-co-glycolic acid) (PLGA). For example, the hollow fibers may include 10% PLGA.

[0104] In some instances, the hollow fibers include a reusable polymer. A reusable polymer refers to a polymer that does not degrade over time or with use in cell culture. A reusable polymer can provide a perfusion module that can be washed and used multiple times. In some instances, the hollow fibers are hydrophobic. In some instances, the hollow fibers are washable. Washable refers to hollow fibers that can be washed and / or sterilized without suffering damage or loss of functionality. Hollow fibers with these properties can reduce waste and costs.

[0105] For example, the hollow fibers may be made from polystyrene.

[0106] Hollow fibers can be solid or semi-solid substrates with openings or apertures (pores) that allow components of cell culture media, such as metabolites, nutrients, and gases (such as oxygen), to be delivered to cells attached to the outer surface of the hollow fiber. The hollow fibers described herein allow cells to grow on the outer surface of the hollow fiber. Thus, the hollow fibers described herein function as a three-dimensional matrix that allows for the culture and maintenance of cells in a three-dimensional structure.

[0107] The hollow fibers may have a Young's modulus of at least 1000 Pa. In some examples, the hollow fibers have a Young's modulus of 1000 Pa to 1,000,000,000 Pa. In some examples, the hollow fibers have a Young's modulus of 8,000 Pa to about 20,000 Pa.

[0108] For example, the hollow fiber may have a Young's modulus of about 115 MPa.

[0109] The pores of the porous polymer sheet may have an average pore size suitable for allowing cell infiltration and support within the polymer sheet. The pores may have an average diameter of at least 0.001 μm to 100 μm. For example, the porous scaffold may have an average pore size of about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.8, 0.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, The pores may have an average diameter of 0.001 μm to 5 μm.

[0110] In some cases, the pores may have an average diameter of about 2.5 μm. In some cases, the pores may have an average diameter of about 5 μm.

[0111] The average pore size can be measured using optical methods such as scanning electron microscopy (SEM), atomic force microscopy (AFM), computed tomography, and / or transmission electron microscopy (TEM). Other methods that can be used include X-ray refractometry, absorption, mercury injection, gas expansion, and the like.

[0112] The average pore size and porosity (or pore density) may affect cell penetration into the scaffold and define the spatial distribution of cells within the 3D matrix of the scaffold. Furthermore, the average pore size and porosity may affect flow resistance, nutrient transport, and / or waste evacuation from cells cultured on and / or within the scaffold.

[0113] The interior lumen of each hollow fiber serves as a conduit for transporting cell culture medium to the cells on the outer surface of the hollow fiber and for transporting waste products from the cells. The pore density of each hollow fiber is at least 1 pore / mm 2 For example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 pores / mm 2 (pores / mm 2 ).

[0114] In some instances, the pore density is about 210 pores / mm 2 There are cases where this can happen.

[0115] The inner lumen of each hollow fiber may have an inner diameter of 1 μm to 1000 μm. The inner diameter refers to the diameter measured between the inner surfaces of the inner lumen. For example, the diameter of the inner lumen may be about 500 μm.

[0116] In some cases, the diameter of the internal cavity is about 600 to about 700 μm. In some cases, the diameter of the internal cavity is about 630 to 660 μm. In some cases, the diameter of the internal cavity is about 50 to about 200 μm.

[0117] Each hollow fiber can have an outer diameter of about 12 μm to 2000 μm.

[0118] In some examples, the outer diameter of each hollow fiber may be 500 to about 1000 μm, for example, 850 to 1000 μm, or about 900 to about 950 μm.

[0119] The length of each hollow fiber may be at least 1 cm. In some cases, the length of each hollow fiber may be up to 10 m. In some cases, the length of each hollow fiber is between 1 cm and 10 m. In some cases, the length of each hollow fiber is between 3 cm and 5 m. In some cases, the length of each hollow fiber is between 10 cm and 2 m.

[0120] Hollow fibers can be made by any method, many of which are known in the art, such as melt spinning, solution spinning, wet spinning, gel spinning, dry-wet spinning, liquid crystal spinning, dispersion spinning, reaction spinning, and electrospinning.

[0121] In some instances, the hollow fibers may be fibers described in Luetchford, Kim A. et al., "Next Generation In Vitro Liver Model Design: Combining Permeable Polystyrene Membranes with Transdifferentiated Cell Lines," Journal of Membrane Science 565 (2018): 425-438, the entire contents of which are incorporated herein.

[0122] In some cases, the perfusion module may include at least one hollow fiber. For example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 150, 200, 250, 300, 400, 500, 600, 800, 1000 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000 or more. In some examples, the perfusion module may include between 1 and 50,000 hollow fibers.

[0123] The methods described herein may include an initial sterilization and / or cleaning step. For example, the perfusion module, tubing, and other components of the perfusion system may be sterilized. Depending on the material of each component, sterilization may be by autoclaving and / or application of a sterilizing composition, e.g., 70% ethanol. For example, a perfusion module without its hollow fibers and reservoirs may be autoclaved before connection to the perfusion system.

[0124] After connecting the perfusion module to the perfusion reactor system, ethanol can be pumped into the system to sterilize the system components. The sterilizing composition can be pumped (or perfused) into the system for at least 30 minutes, e.g., at least 30, 40, 50, 60 minutes, or more.

[0125] After sterilization, the method may include a cleaning step. The cleaning step may include removing any sterilizing composition from the system, for example, by draining the sterilizing solution from the system. The cleaning step may further include pumping culture medium through the system to saturate the system and contact all components with the culture medium. The cell culture may then be maintained in the system. In some cases, the culture medium used in the cleaning step may be replaced with additional culture medium as described herein, for example, a growth medium as described herein.

[0126] The methods described herein include seeding cells onto hollow fibers in a perfusion module. Prior to seeding, the cells may be pre-cultured. For example, cells may be pre-cultured outside the perfusion system to form a cell suspension (seeding culture).

[0127] A cell suspension may be formed by first washing the cells with a buffer solution such as phosphate-buffered saline (PBS). The buffer solution may contain additional components, such as a cell detachment agent. The cell detachment agent may be an enzyme, such as trypsin, trypLE, or nattokinase. In some cases, the cell detachment agent may be a composition, such as actuase. For example, the buffer solution may contain at least 0.05% trypsin. In some cases, the buffer solution may contain about 0.25% trypsin. Additional components may then be added to the buffer solution containing the cells. For example, EDTA may be added to the buffer solution and the cells. The buffer solution and the cells may then be incubated for a period of time, such as at least 3 minutes, or for about 5 minutes.

[0128] After incubation, a portion of the cells in the buffer solution can be mixed with culture medium containing a growth promoter, such as serum, e.g., fetal bovine serum (FBS). Serum may help neutralize the cell detachment agent. Other examples of growth promoters include growth factors (e.g., recombinant growth factors), bovine eye fluid, sericin protein, and heat-inactivated earthworm coelomic fluid. In some cases, the culture medium is serum-free and may contain additional components as needed, depending on the cells being cultured. For example, serum-free media include those commercially available from ThermoFisher, Lonza Bioscience, and Merck. The number of cells in a given volume of culture medium can then be determined, e.g., using a cell counter or other appropriate method.

[0129] After determining the number of cells in a given volume of culture medium, the volume of culture medium can be centrifuged to pellet the cells. The cell pellet can then be resuspended in culture medium, such as a growth medium described herein, to form a cell suspension.

[0130] The cell suspension can then be applied to the perfusion module and the hollow fibers therein. In some cases, the volume applied to the perfusion module can be a volume that at least partially fills the extracapillary space (ECS). The remaining volume of the perfusion module can be filled with additional culture medium (without cells). In some cases, the volume of the cell suspension can be less than the ECS. In some cases, the cell suspension can be continuously circulated through the perfusion module and thus continuously contact the hollow fibers. Continuous circulation of the cell suspension can increase seeding efficiency.

[0131] In some cases, the number of cells applied to the perfusion module can be determined by the external surface area of ​​the hollow fibers, e.g., at least about 1000 cells / cm. 2 may be applied to the perfusion module. In some instances, about 25,000 cells / cm 2may be applied to the perfusion module. For example, cells may be placed in each square centimeter of each hollow fiber at a density of at least 100 cells / cm. 2 Cells may be added at a concentration of 100 cells / cm for each square centimeter of external surface area of ​​all hollow fibers. 2 up to 500,000 cells / cm 2 For example, at least 100 cells / cm 2 , 200 cells / cm 2 , 300 cells / cm 2 , 400 cells / cm 2 , 500 cells / cm 2 , 600 cells / cm 2 , 700 cells / cm 2 , 800 cells / cm 2 , 900 cells / cm 2 , 1000 cells / cm 2 , 1500 cells / cm 2 , 2000 cells / cm 2 , 2500 cells / cm 2 , 3000 cells / cm 2 , 3500 cells / cm 2 , 4000 cells / cm 2 , 4500 cells / cm 2 , 5000 cells / cm 2 , 5500 cells / cm 2 , 6000 cells / cm 2 , 6500 cells / cm 2 , 7000 cells / cm 2 , 7500 cells / cm 2 , 8000 cells / cm 2 , 8500 cells / cm 2 ,9000 cells / cm 2 , 9500 cells / cm 2 , 10000 cells / cm 2 , 10500 cells / cm 2 , 11000 cells / cm 2 , 11500 cells / cm 2 , 12000 cells / cm 2 , 12500 cells / cm 2 , 13000 cells / cm 2 , 13500 cells / cm 2 , 14000 cells / cm2 , 14500 cells / cm 2 15,000 cells / cm 2 15500 cells / cm 2 , 16,000 cells / cm 2 , 16500 cells / cm 2 , 17,000 cells / cm 2 , 17500 cells / cm 2 , 18,000 cells / cm 2 , 18500 cells / cm 2 , 19,000 cells / cm 2 , 19500 cells / cm 2 20,000 cells / cm 2 20500 cells / cm 2 21,000 cells / cm 2 21500 cells / cm 2 22,000 cells / cm 2 22500 cells / cm 2 23,000 cells / cm 2 23500 cells / cm 2 24,000 cells / cm 2 , 24500 cells / cm 2 25,000 cells / cm 2 25500 cells / cm 2 26,000 cells / cm 2 26500 cells / cm 2 27,000 cells / cm 2 27500 cells / cm 2 28,000 cells / cm 2 , 28500 cells / cm 2 29,000 cells / cm 2 , 29500 cells / cm 2 30,000 cells / cm 2 30500 cells / cm 2 31,000 cells / cm 2 31500 cells / cm 2 32,000 cells / cm 2 32500 cells / cm 2 33,000 cells / cm 2 33500 cells / cm 2 34,000 cells / cm 2 34,500 cells / cm 235,000 cells / cm 2 35500 cells / cm 2 36,000 cells / cm 2 36,500 cells / cm 2 37,000 cells / cm 2 37,500 cells / cm 2 38,000 cells / cm 2 38,500 cells / cm 2 39,000 cells / cm 2 39500 cells / cm 2 40,000 cells / cm 2 40500 cells / cm 2 41,000 cells / cm 2 41500 cells / cm 2 42,000 cells / cm 2 42500 cells / cm 2 43,000 cells / cm 2 43500 cells / cm 2 44,000 cells / cm 2 44,500 cells / cm 2 45,000 cells / cm 2 45,500 cells / cm 2 46,000 cells / cm 2 46,500 cells / cm 2 47,000 cells / cm 2 47,500 cells / cm 2 48,000 cells / cm 2 48,500 cells / cm 2 49,000 cells / cm 2 49,500 cells / cm 2 50,000 cells / cm 2 50500 cells / cm 2 , 51,000 cells / cm 2 51500 cells / cm 2 , 52,000 cells / cm 2 , 52500 cells / cm 2 , 53,000 cells / cm 2 , 53500 cells / cm 2 , 54,000 cells / cm 2 , 54500 cells / cm 2 55,000 cells / cm 2 55500 cells / cm2 , 56000 cells / cm 2 , 56500 cells / cm 2 , 57000 cells / cm 2 , 57500 cells / cm 2 , 58000 cells / cm 2 , 58500 cells / cm 2 , 59000 cells / cm 2 , 59500 cells / cm 2 ,60000 cells / cm 2 ,70000 cells / cm 2 , 80000 cells / cm 2 ,90000 cells / cm 2 , or 100,000 cells / cm 2 , 200000 cells / cm 2 , 300000 cells / cm 2 ,400000 cells / cm 2 ,500000 cells / cm 2 , 1,000,000 cells / cm 2 Or even more.

[0132] In some cases, the number of cells may be determined based on the number of cells used or the number of cells seeded into a 2D cell culture system. For example, the number of cells applied may be at least 0.1 times greater than the number used to seed cells into a planar sheet (2D) cell culture system made of the same material as the hollow fibers. For example, the number of cells applied may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 times greater than the number used to seed cells into a planar sheet (2D) cell culture system made of the same material as the hollow fibers.

[0133] After the cells are applied to the perfusion module, the perfusion module can be maintained at an angle of 0 to 90 degrees from horizontal for a period of time sufficient for the cells to attach or adhere to the outer surface of the hollow fibers. Maintaining the perfusion module at an angle as described herein allows for initial attachment of the cells and avoids uneven distribution of the cells on the outer surface of the hollow fibers.

[0134] In some examples, the angle is about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°.

[0135] In some cases, the module is maintained at an angle of 0 to 30 degrees from the horizontal. For example, 5 to 20 degrees from the horizontal. For example, 8 to 10 degrees from the horizontal. For example, 8.5 to 9.5 degrees from the horizontal. In some cases, the angle is approximately 9.2 degrees. That is, the hollow fibers within the module are maintained at a selected angle. The hollow fibers may be of any shape, and the angle may be defined relative to an axis passing through the hollow fibers. For example, in the case of tubular, substantially straight hollow fibers, the angle from the horizontal may be taken as the angle between the horizontal plane and an axis passing longitudinally through the hollow fibers (i.e., the axis is the same as the internal lumen of the hollow fibers). In some cases, the hollow fibers may be helical, arcuate, or non-linear. In such cases, the angle may be defined by an offset from a plane approximately parallel to the horizontal. That is, when the perfusion module is placed in a horizontal plane, the perfusion module is offset relative to the horizontal plane, and therefore the fibers therein are offset from the horizontal plane. It will be understood that hollow fibers may not be uniform in shape, and therefore references to angles from the horizontal refer to angles taken from a particular point on the perfusion module or hollow fiber (i.e., the center point of the vertical axis through the perfusion module or hollow fiber), and therefore the angle may not be the same for all portions of the hollow fiber.

[0136] In some cases, the perfusion module may be maintained at an angle described herein for at least 10 minutes, and in some cases, the perfusion module may be maintained at that angle for the entire incubation period and / or culture (proliferation and / or differentiation).

[0137] After maintaining the perfusion module, the perfusion module, including the cells seeded therein, may be incubated for a period of time to allow further cell attachment and initial growth. The perfusion module and cells may be incubated for at least 2 hours, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 hours, or more. In some cases, the perfusion module and cells may be incubated for approximately 3 hours. The perfusion module and cells may be incubated under conditions (i.e., temperature and atmosphere) suitable for maintaining viable cells and / or allowing cell growth. For example, in the case of mammalian cells, the perfusion module and cells may be incubated at 37°C in an atmosphere containing 5% CO2.

[0138] The perfusion module can be rotated during the initial attachment phase (seeding) and / or incubation period. In some cases, the perfusion module and cells rotate intermittently. In some cases, the perfusion module and cells rotate at a speed of 0-30 rpm. In some cases, the perfusion module and cells rotate at a speed of about 12 rpm. In some cases, the perfusion module and cells rotate every 10 seconds to 30 minutes for 1 second to about 30 minutes. In some cases, the perfusion module and cells rotate for about 30 seconds every 5 minutes. That is, the perfusion module rotates for 30 seconds, then remains stationary for 5 minutes, and then rotates again. The perfusion module can rotate for at least 1 hour, e.g., at least 1, 2, 3, 4, or 5 hours.

[0139] In some cases, the perfusion module may rotate continuously. The perfusion module may rotate continuously at a speed of 0 to 30 rpm. In some cases, the perfusion module rotates continuously at a speed of 0 to 4 rpm. In some cases, the perfusion module rotates continuously for at least 3 hours, for example, about 3.5 to about 5 hours.

[0140] Rotation of the perfusion module and cells can be accomplished by any suitable means, for example using a tube rotator such as the MACSmix™ available from MiltenyiBiotech.

[0141] Rotation of the perfusion module and cells can be used to apply centrifugal force to the cells. Therefore, the centrifugal force can be greater than 0.001 N. For example, the centrifugal force can be at least 0.001 N. In some cases, the centrifugal force can be between 0 and 50 N. The application of centrifugal force can help move the cells through the perfusion module and bring them into contact with the hollow fibers. Without being bound by theory, this can help improve the efficiency of cell attachment to the hollow fibers. Intermittent rotation of the module can help introduce a driving centrifugal force to the suspended cells, potentially improving mixing. Increased mixing can help distribute the cells evenly across the hollow fiber surface upon contact and attachment.

[0142] The force exerted by rotation can be influenced by the orientation of the perfusion module relative to the axis of rotation. For example, the perfusion module can be positioned so that there is an offset between the midpoint of the axial centerline of the perfusion module and the axis of rotation. The centerline of the perfusion module refers to a plane that passes axially longitudinally through the center of the perfusion module, as indicated by the cross in Figure 6. The midpoint of the centerline refers to the midpoint along the centerline. Providing an offset in combination with the rotation described above may help mix cells within the perfusion module. Without being bound by theory, the mixing provided by the offset and rotation may increase the surface area of ​​the hollow fibers that cells come into contact with, thus improving the efficiency of cell attachment to the hollow fibers.

[0143] Maintaining the perfusion module and / or rotation (intermittent or continuous) can help improve cell attachment and / or increase the number of viable cells (i.e., viable cells attached to the hollow fibers), for example, by increasing attachment efficiency by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more compared to methods that do not include the maintenance steps and / or rotation described herein.

[0144] Cell viability can be measured using any suitable means known in the art. For example, cell viability can be measured by a direct measurement method. A direct measurement method can include attaching cells to hollow fibers. Unattached cells are then discarded, for example, by flushing the perfusion module with additional culture medium. Prior to culturing (i.e., proliferation or differentiation), the fibers containing the attached cells are removed, and the cells attached to the fibers are removed (i.e., detached) from the fibers by known means, for example, using a detaching agent. The number of viable cells detached from the fibers can then be determined. The number of viable cells can be determined by a cell counting method, such as flow cytometry or FACS, for example, using a cell counter such as the Chemometec NC-200. The total number of attached cells and / or the total number of viable cells can be used to determine attachment efficiency.

[0145] In some cases, cell viability after attachment can be measured by an indirect method. For example, the growth medium is discharged from the perfusion module, and the total number of cells in the discharged growth medium (including non-viable and viable cells) is quantified using the cell counting method described above. The attachment efficiency is then calculated using the following formula: indirect attachment efficiency = (inoculated cells - cells removed during discharge after seeding) / inoculated cells (%).

[0146] After rotating the perfusion module and cells, the perfusion module is connected to the perfusion system, for example, by connecting tubing to both ends of the perfusion module and any side ports that may be included.

[0147] The cell culture medium described herein can then be perfused (pumped) through the perfusion system and the internal lumen of the hollow fibers. The cell culture medium can be a growth medium described herein. The culture medium can be pumped from either end of the perfusion module. In some examples, the perfusion module is maintained at an angle between 0° and 90°. For example, the perfusion module can be at an angle of about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90°. Thus, culture medium can be pumped from the bottom or top end to the opposite end. In some instances, culturing involves a perfusion module and maintaining the hollow fibers with attached cells at a 0° angle from horizontal.

[0148] Cells can be cultured under conditions suitable for cell growth. For example, the conditions described herein can be used, but can be selected depending on the type of cells to be cultured. For example, mammalian cells can be cultured (incubated) at 37°C in an atmosphere containing 5% CO2.

[0149] In some instances, the culture medium may be changed to a second culture medium. For example, the culture medium may be changed from a proliferation medium to a differentiation medium as described herein. After culturing in the differentiation medium, the cells become striations. The cells can form myotubes. The cells can form striated myotubes along each channel. Without being bound by theory, mechanical forces due to shear stress generated by fluid flowing through the ECS and / or the internal lumen may help the cells to form striations.

[0150] The second culture medium may be changed to an additional medium, which may be the same as the initial culture medium (e.g., switching from a differentiation medium to a proliferation medium) or may be a different culture medium.

[0151] The culture medium can be switched any number of times from the first medium, the second medium, or subsequent mediums. In some instances, the culture conditions may be changed depending on the medium perfused through the system.

[0152] The cells can be cultured in any of the first, second, and third culture media for at least 3 hours. In some cases, the cells can be cultured for at least 1 day, for example, 1, 2, 3, 4, 5, 6 days, or more. In some cases, the cells are cultured continuously. This can provide a system that can provide a large number of cells.

[0153] After culturing the cells, the cells can be harvested from the perfusion module. The cells can be harvested by removing the cells from the hollow fibers. In some cases, if the hollow fibers are formed from an edible, biodegradable, or digestible polymer, the cells can remain attached to the hollow fibers and be harvested, for example, by removing the hollow fibers with attached cells from the perfusion module.

[0154] The cells can be harvested when they reach at least 60% confluence. In some cases, the cells can be harvested after proliferation and / or differentiation, for example, after culturing in a growth medium for a period of time as described herein. If the culture includes the use of a differentiation medium, the cells can be harvested when the cells reach a predetermined protein density.

[0155] The harvested cells, or the harvested cells and hollow fibers, may be used to produce edible products, for example, the cells and hollow fibers may be formed into a meat analog.

[0156] Meat analogs (also known as cultured or in vitro meat) refer to foods that are not produced by the slaughter of animals but have the same or similar structure, texture, aesthetic qualities, and other characteristics as meat from slaughtered animals, including livestock (e.g., beef, pork), game (e.g., venison), poultry (e.g., chicken, turkey, duck), and / or fish or seafood substitutes / analogs. The term refers to raw, cooked, and prepared meat-like foods.

[0157] The cells or cells and hollow fibers may be configured to mimic the taste, texture, size, shape, and / or structure of traditional slaughtered meat. For example, multiple sets of harvested cells or cells and hollow fibers may be combined, e.g., bonded together or pressed together, to form a structure similar to a cut of meat or portion-sized product. For example, the harvested cells or cells and hollow fibers may be bonded together by an edible adhesive agent, such as transglutaminase.

[0158] Additional agents may be added to the harvested cells or cells and hollow fibers to more closely resemble meat in sensory properties such as texture, taste, odor, and visual characteristics. For example, one or more of fats, texture modifiers, bulking agents, thickeners, preservatives, flavor enhancers, antimicrobial agents, pH adjusters, desiccants, vitamins, minerals, metals, salts, sweeteners, salting or pickling agents, colorants, or any combination thereof may be added to the cells or cells and hollow fibers. The additional agents may be dispersed into the cells or cells and hollow fibers via the internal lumen of the hollow fibers.

[0159] Accordingly, also provided herein is a meat analog comprising cells or cells and hollow fibers produced by the methods described herein.

[0160] The meat analogues produced may have the dimensions of whole meat, for example, at least one dimension (i.e., at least one of length, width, or thickness) may be 10 cm or greater. The thickness or diameter of such meat analogues may be up to 50 cm.

[0161] If the hollow fibers are not edible, cells can be removed from the hollow fibers. That is, cells can be recovered from the hollow fibers. Cells can be recovered by applying a cell-support-specific agent to the hollow fibers. The cell detachment agent can be any agent capable of detaching cells from the hollow fibers. For example, the cell detachment agent can be an enzyme, such as trypsin, trypLE, or nattokinase. In a specific example, the cell detachment agent is nattokinase. Nattokinase is a fibrin-specific enzyme derived from fermented soybeans. The nattokinase can be food-grade nattokinase.

[0162] The cell detachment agent may be added to the hollow fibers, for example, after they have been spread into a flat sheet, or may be applied to the hollow fibers as is. The cell detachment agent may be applied at a concentration of at least 10 mg / ml. For example, the cell detachment agent may be applied at a concentration of at least 10, 20, 30, 40, 50, 60, or 70 mg / ml.

[0163] After application of the cell detachment agent, the cells are removed from the hollow fibers and suspended in a composition containing the cell detachment agent, and the recovered cells can be separated from the composition by, for example, centrifugation or other known methods.

[0164] The recovered cells can then be used to produce edible products. The cells can be processed into products such as meat analogs. For example, the cells can be subjected to processes similar to those used to produce products such as sausages or processed meat products, e.g., restructured meats such as baloney. For example, the cells can be emulsified, ground, or minced, and then formed into products resembling meat cuts or meat products. For example, the processed cells can be molded or shaped using any known method. Agents such as fats, binders, or texture modifiers can be added to the cells to aid in processing. Additional agents can be added to the cells to more closely resemble meat in sensory properties, such as texture, taste, odor, and visual characteristics. For example, one or more of fats, texture modifiers, bulking agents, thickeners, preservatives, flavor enhancers, antimicrobial agents, pH adjusters, desiccants, vitamins, minerals, sweeteners, salts, metals, curing or pickling agents, colorants, or any combination thereof can be added to the cells.

[0165] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2nd Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used to practice the present invention, preferred methods and materials are described herein. Therefore, the terms defined below are more fully explained by reference to the entire specification. Additionally, the singular terms "a," "an," and "the" used herein include plural references unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that the present invention is not limited to the particular methodology, protocols, and reagents described, which may vary depending on the circumstances employed by those of skill in the art.

[0166] Aspects of the present invention are demonstrated by the following non-limiting examples. <Example>

[0167] Example 1 - HFB setup for muscle cell culture 1. Fiber 1.98 Fibers are produced by the phase-inversion spin-casting method, the details of which are given in [4] and [5]. NOTE: In this work, fibers were fabricated in-house using a non-biodegradable proprietary polymer, NMP as the solvent, and HO as the non-solvent. The fibers used in the system described here have an outer diameter of 1.05 mm and an inner lumen diameter of 600–700 μm. The fibers are porous, with a pore size of 2.28 μm ± 1.5 μm (mean ± standard deviation). This is designed to separate cells from the media supply within the fiber inner lumen, recapitulating the vasculature of tissue. Fibers can also be purchased from membrane suppliers such as Pall.

[0168] 2. Module manufacturing NOTE: The module used in this study is made of 1 mm thick borosilicate glass with two side ports (Figure 2A). The fiber in the module described here consists of three fibers, with a total external surface area of ​​6.54 cm. 2 This corresponds to approximately half of a well in a 6-well plate. 2.1. Before first use, coat the inner surface of the module with Sigmacote, dry it in a fume hood and siliconize it. To extend the duration of the treatment, autoclave it (121°C, 1 atmosphere, 20 minutes). 2.2. Cut 75 mm long fibers using a scalpel and insert three fibers into each module, leaving approximately 7 mm (~7 mm) of excess length on both ends (Figure 3A). 3.3. Place approximately 0.5 mL (~0.5 ml) of silicone adhesive into a weigh boat. Use a P200 pipette tip to pick up a small amount of silicone and apply it around the fibers at the edge of the module, forming a 3-5 mm plug (Figure 3B). Allow to dry for at least 3 hours. 3.4. Using a scalpel, cut the silicone flush with the edge of the glass module (Figure 3C). 3.5. Wrap a small amount (up to about 4 layers) of polytetrafluoroethylene (PTFE) tape around the port on one side.

[0169] 3. System Setup and Sterilization NOTE: The pump tubing and fibered module are not autoclaved and are sterilized using 70% ethanol. It is recommended to calibrate the pump tubing to match the pump being used. The following steps are performed in a laminar flow hood. 3.1. Prior to setup, autoclave all autoclavable components (see section 2.1). Setup 3.2.1. Pour 10 ml of 70% ethanol into the reservoir bottle and set up the reservoir bottle, Q series cap, feed tube, pump, and pump tube as shown in Figure 4. 3.2.2. Loosely install an end cap onto the PTFE-taped side port. Slide the end of the L / S16 module connector onto the end of the module and into the open side port. Connect a 40 mm section of L / S13 tubing to the module connector closest to the capped side port, as shown in Figure 2B. 3.2.3. Connect the module to the pump tubing and orient the module so that the capped side port is closest to the pump. 3.2.4. Connect the permeate and retentate lines to the module connector and L / S14 of the reservoir bottle Y-connector. Ensure the setup resembles the schematic in Figure 4. 3.3. Sterilization 3.3.1. Pump ethanol through the module at 800 μl / hr (267 μl / hr per fiber) or 30 ml / hr for a time sufficient to process non-autoclaved parts for at least 30 minutes (e.g., 1 hour) (adjust time if using other sterilization methods [6]). 3.4. Cleaning 3.4.1. To flush ethanol from the system, first turn off the pump and drain the tubing. First, remove the pump tubing from the module adapter tubing. Lift the module and drain the ethanol from the fibers and retentate lines back into the reservoir bottle. Remove the side port end cap from the module and drain the ethanol from the module itself and the permeate lines. Reinstall the side port end cap. Disconnect the inlet pipe to the reactor and place it in an empty container, then turn on the pump to empty the lines between the reservoir and reactor (now empty). Disconnect the inlet pipe to the reactor and place it in an empty container, then turn on the pump to empty the lines between the reservoir and reactor (now empty). Alternatively, reverse the medium flow on the pump to drain the pump tubing and the ethanol supply lines. Turn off the pump and reattach the pump tubing to the module adapter. 3.4.2. Remove the cap from the ethanol bottle and replace it with a bottle containing 10 ml of serum-free cell growth medium (EMEM, GMEM, DMEM, RPMI, etc.) (e.g., run DMEM (serum-free) at 30 ml / h for 1 hour). Pump medium through the system until the retentate line is filled with medium. Clamp the retentate line to allow the medium to permeate the fibers and wash the module. Allow to wash for approximately 2 hours.

[0170] Next, perform a preconditioning step using growth medium (DMEM + 10% FBS + 1% P / S). Replace the wash medium with a reservoir containing 10 ml of growth medium (DMEM + 10% FBS + 1% P / S) and pump at 30 ml / h overnight. Replace the wash medium with a reservoir containing 10 ml of growth medium (DMEM + 10% FBS + 1% P / S) and pump at 30 ml / h overnight.

[0171] 4. Seeding NOTE: The medium and supplements used in this protocol should be those established for the cell type of interest. For details, refer to the literature, the European Collection of Cell Cultures (ECACC), and the American Type Culture Collection (ATCC). Before using this method, cells should be maintained according to established protocols for the cell type of interest. In this work, C2C12 cells were used and maintained according to the distributor's recommendations (ATCC). NOTE: For C2C12 cells, a separate pretreatment step may be performed. The seeding protocol presented below is also useful for pre-incubating the module with cell culture medium prior to cell growth. If a larger pre-culture is required, the pre-incubation should be performed before seeding the module by draining the wash medium from the system, replacing it with growth medium, and allowing it to permeate the module for several hours. For details on changing the medium in the system, see section 7.5.2.1. 4.1 Prepare a single cell suspension by trypsinization according to the protocol specified for the cell type of interest. A typical protocol for T75 culture is as follows: NOTE: The number of cells to use in the seeding step needs to be empirically determined depending on the cell type of interest. The bioreactor described here is seeded at a cell density that, for C2C12 cells, is four times the cell density used on 2D tissue culture plastic for 7-day culture. 4.1.1. Wash the cells by adding 10 ml of phosphate-buffered saline (PBS), aspirating, then adding 3 ml of 0.25% trypsin and ethylenediaminetetraacetic acid (EDTA) (enough to cover the cells) and incubating at 37°C, 5% CO2 for 5 minutes. Harvest the cells in 7 ml of culture medium supplemented with 10% fetal bovine serum (FBS) to neutralize the trypsin. Mix well and add 10 μl to the chamber of a hemocytometer to count the cells. Use a Chemometec NC-200 cell counter for cell counting. 4.1.3. Pellet the cells by centrifugation at 200 x g for 5 minutes. 4.1.4. Aspirate the supernatant and resuspend the C2C12 cell pellet (<80% confluence) obtained from the T-75 flask in 2 ml of medium prior to cell counting to ensure a high starting cell concentration. 4.2. Turn off the pump and drain the feed tube and module as in 3.4.1. 4.3. Remove the module from the module connector and attach a module end cap (Table 1) pre-sterilized with 70% ethanol, leaving one side port free. 4.4. 160,500 cells (1.605 × 10 5 200 ul of cells (inoculation density 25,000 cells / cm) was accurately transferred to the module. 2 Next, add additional growth medium (without cells) to fill the remaining space in the ECS of the module. NOTE: It is important to use 200 µL in this example because using 500 µL will result in insufficient volume of ECS and not all of the cell suspension will be transferred to the reactor. 4.1.1 Cap the side port using an end cap. Incubate the cells at 37°C, 5% CO2 for 3 hours. After adding the cell suspension, the reactor module must be oriented and maintained at a predetermined angle to prevent uneven distribution of cells due to gravity settling. The module must be maintained at the selected angle for at least 10 minutes (specific to the initial mounting time of the C2C12s) before mounting on the tube rotator. 4.1.2. Using a tube rotator (Miltenyi Biotech MACSmixl), rotate the module intermittently at 12 rpm for 30 seconds with a 5 minute rest between rotation periods. 4.5. The module can be drained by introducing air using a 27G needle or by removing the side port end cap and allowing it to drain by gravity. After seeding, attach an end cap to the injection port (sterilized with 70% ethanol) and attach it to the side port (secured with PTFE tape). Remove the end cap from the other side port and use a 27G needle and 1 ml syringe to inject air into the attached injection port to slowly expel the cells. 4.5.2. Replace the injection port with an end cap. Using the free side port and an 18G needle, 1 ml syringe, slowly fill the module with medium. Remove the end cap of the module and connect the module to the tubing using the module connector. 4.6. Replace the wash medium bottle with a bottle containing 8.5 mL of growth medium. Pump the growth medium into the system. NOTE: If differentiation is required, replace with a bottle containing differentiation medium (DMEM + 2% horse serum + 1% P / S).

[0172] 5. Proliferation NOTE: Continuous monitoring of dissolved oxygen concentrations in the reactor inlet and outlet streams is performed to provide online data on the growth of C2C12 cells. NOTE: The fibers used in the research system described here are set to permeate at approximately 80 μl / hr (~80 μl / hr) with a feed rate of 800 μl / hr. 5.1. Culture cells with the fibers in a humidified incubator set at 37°C and 5% CO2 for up to 6 days. NOTE: Monitoring nutrients and metabolites during the growth phase provides useful information about cell growth, metabolic uptake and output, and nutrient and metabolite levels in the medium. For example, glucose utilization and lactate production. Using a metabolic analyzer (Roche CEDEX), glucose, lactate, glutamine, and ammonia concentrations can be measured. Kits capable of quantifying these factors from the medium are available from various suppliers (see Table 1 for the kits used in this study). Injection ports were added to the permeate and retentate tubing, allowing medium sampling with a 27G needle and syringe. Alternatively, medium could be sampled from the medium reservoir bottle. This provides nutrient and metabolic information for both the input and output streams. Sampling should be performed in a laminar flow hood. Prior to sampling, sterilize the injection ports by pressing a blue roll soaked in ethanol against the ports for at least 30 seconds.

[0173] 6. Excision NOTE: At the end of the experiment, the fibers can be excised from the module for analysis. 6.1. Unplug and drain the HFB. 6.2. Insert a scalpel / micro knife blade between the glass and silicone. Rotate the module to cut the silicone from the glass. Repeat this procedure on both ends of the module. 6.3. Use a blade to hook one end of the silicone plug and pull gently, making sure the fibers are attached as well.

[0174] 7. Cell analysis 7.1. Cell number NOTE: For the C2C12 cells used in this study, any time point within the 6-day growth period is suitable for this calculation, as growth rates do not change significantly at cell densities achieved within this period. 7.1.1. After excision (section 6), wash the fibers by immersing them in PBS and place them in a 1.5 ml tube containing 0.5 ml Tris-EDTA (TE) buffer. 7.1.2. Cut the fiber into three axial sections (to represent the inlet, middle, and outlet sections) 7.1.3. Add trypsin (0.25%) to 1 ml and place in the incubator for 5 minutes. 7.1.4. Next, vigorously pipette to separate the cells from the fibers and break up any cell aggregates. 7.1.5. Next, take a cell sample by placing a ChemomeTec NC-200 cassette into the suspension and perform a cell count and cell viability check using a ChemomeTec NC-200 cell counter. 7.2. Cell proliferation rate 7.2.1. Using the calculated cell numbers at the two different time points, calculate the specific growth rate, p (Equation 1), where Ln(X1) is the natural logarithm of the cell number at the first time point and Ln(X2) is the natural logarithm of the cell number at the second time point. Equation 1: p=(Ln(X2)-Ln(X1)) / time(hr) From this, the population doubling time (dT) is calculated (Equation 2), where μ is the specific growth rate. Equation 2: dT=Ln2 / μ 7.3. Cell viability 7.3.1 After excision (section 6), wash the fibers in PBS and place them in a 1.5 ml tube containing 500 μl of 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA). Incubate at 37°C for 10 minutes. 7.3.2. Add 10 μl of cell suspension to 10 μl of trypan blue and mix. Load 10 μl onto a hemocytometer and count the number of dead (blue) and viable cells. 7.4. Imaging 7.4.1 Wash the excised fibers by immersing them in PBS, cut them into short lengths with scissors, and place them in a 24-well plate. Add 400 μl of 4% paraformaldehyde in PBS and incubate at room temperature for 20 minutes. 7.4.2. Wash with PBS by pipetting 400 μL each. Repeat this step with fresh PBS. 7.4.3. Add 400 μl of 4',6-diamidino-2-phenylindole (DAPI) diluted approximately 3:3 in PBS to 100 ng / ml and incubate at room temperature for 20 minutes. Avoid light. 7.4.4. Wash twice with PBS (as in 7.3.2) and once with HO. Add fluorescent mounting medium to cover the fibers and image immediately to collect data (DAPIex / em; 359 / 461 nm) before the sample dries. Take images at different focal planes and use "focus stacking" software (such as the Stack Focuser plugin from Imaged below) to create a composite image with a significantly increased depth of field. This is necessary because the fibers are not flat. 7.4.5.1. Download Imaged (http: / / imaged.nih.gov / ij / ) and the "stack-focuser" plugin (http: / / rsb.info.nih.gov / ij / plugins / stack-focuser.html). 7.4.5.2. Open the images to be stacked in Imaged. Then, go to [Image] menu, [Stacks], [Images to be Stacked]. Go to [Plugins] menu, and [StackFocuser]. Specify n for the nxn kernel. Trial and error with n may be required to produce an image with less noise. Values ​​between 11 and 77 tend to work well.

[0175] Example 2 - Hollow fiber bioreactor for cell-based meat production Materials and Methods Cell culture was performed using the immortalized mouse myoblast cell line C2C12 as a model skeletal muscle cell line and primary adult rat skeletal muscle cells RSkMC (Sigma-Aldrich, R150-05a). C2C12s were cultured in DMEM (Sigma-Aldrich, D5796) supplemented with 10% v / v fetal bovine serum (FBS) (Fisher Scientific Ltd, 11573397) and 1% v / v penicillin / streptomycin P / S (5,000 U penicillin and 5 mg / mL streptomycin, Sigma-Aldrich, P4458). RSkMCs were cultured in rat skeletal muscle cell growth medium (Sigma-Aldrich, R151-500). During culture and bioreactor run, cells were incubated at 5% CO2 and 37°C. Cell viability was assessed using trypan blue exclusion method using 0.4% or 0.04% trypan blue solution (Sigma-Aldrich, T8154) and hemocytometer cell count.

[0176] The hollow fiber bioreactor system consisted of a Watson-Marlowe 205U peristaltic pump with orange and white color-coded pump tubing, silicone tubing (0.8 mm inner diameter, L / S13 Platinum-Cured, Masterflex 96410), and a feed bottle with a Whatman Hepta-vent filter unit (Sigma-Aldrich, WHA67235000). The bioreactor module was a custom-built glass module with two inlets / outlets and two side ports (Soham Scientific), measuring 3 mm inner diameter, 5 mm outer diameter, and 60 mm long. System optimization involved the use of an inline dissolved oxygen sensor (PreSens Flow-Through Cell for Oxygen, FTC-SU-PST3-S) coupled with a PreSens Fibox4 oxygen reader. The hollow fibers used in the system were initially 10% PLGA, but later replaced the porous hydrophilic polystyrene hollow fibers developed by Luetchford et al. (2018) and manufactured in-house by wet-dry spinning. [1] The transition to polystyrene hollow fibers, called PX40, was made after the biocompatibility of RSkMCs was tested on flat membranes and compared to a tissue culture plastic control, TCP, using a resazurin-based assay to determine metabolic activity as a surrogate for cell proliferation. Reactors were used with two or three hollow fibers and a total feed flow rate of 800 μl / h (μl h-1). Reactor setup and operation were performed as described by Storm et al. (2016), with a 3-hour attachment period and dynamic seeding using a MACSMix rotator. [2]

[0177] (result) The results presented here summarize the work carried out in this study. First, the suitability of a porous hydrophilic polystyrene membrane (PX40) as a scaffold for cell proliferation was evaluated in terms of biocompatibility and its ability to sustain viable cell growth. This was performed on RSkMCs and compared to tissue culture plastic (TCP) as a positive control, as shown in Figure 5.

[0178] The results of some bioreactor experiments are shown in Table 1. The starting number of cells attached to the hollow fibers on day 0, NO, is assumed to be 10% of the inoculum due to the cell seeding efficiency on the hollow fibers. This efficiency is based on previous work by the Ellis group on the human osteosarcoma (bone) cell line MG-63s. [3] [Table 1]

[0179] (Consideration) Initial biocompatibility testing of RSkMCs on 2D flat sheet membranes (Figure 5) determined that polystyrene membranes were suitable for use as scaffolds. Figure 5 demonstrates that the cells were metabolically active and, consequently, able to attach and grow on the scaffolds. Polystyrene is neither edible nor biodegradable; therefore, polystyrene scaffolds could potentially be reused for multiple batches to minimize operational costs associated with skeletal muscle cell growth.

[0180] The initial reactor studies without unexpected equipment failure and resulting in successful cell perfusion for the desired period were reactor runs 3, 4.1, and 4.2. Assuming a 10% seeding efficiency for the hollow fibers, Table 1 shows that cell proliferation occurred in all reactor runs, with NF > NO. After each of these runs, viability assessment using trypan blue exclusion indicated that all cells were nonviable. These are likely false negatives due to the sensitivity of the primary cell line RSkMC to trypan blue or the effects of exposure to the dissociation enzyme trypsin. The suspected cause of the false identification of nonviable cells due to overexposure to trypan blue could be the high concentration (0.4%) or the exposure period before cell counting, which supports the indeterminate cell color during analysis.

[0181] The number of cells obtained from a bioreactor system can theoretically be increased by increasing the duration of the expansion phase, the reactor run time, and the number of hollow fibers in the reactor module. The number of hollow fibers determines the surface area available for cell attachment and growth, while the confluence determines the need for transfer to a larger reactor system, known as subculture. NF values ​​likely underestimate the final total cell count due to the presence of large cell aggregates during hemocytometer counting. The presence of aggregates is likely due to the method of cell dissociation from the fiber scaffold (trypsin, as currently used).

[0182] Future studies will expand RSkMCs in a bioreactor system equipped with equipment for online monitoring to confirm viability. As an alternative to manual cell counting, DNA quantification methods such as the PicoGreen assay will be used to quantify cell numbers. Other potential approaches include scaling up expansion by extending the expansion period, incorporating more fibers to increase the available scaffold surface area, using larger reactor modules, or operating multiple HFBs in parallel to increase numbers.

[0183] (Conclusion) This study fulfilled the original objective of this study and provides proof-of-concept for the use of hollow fibers, specifically porous polystyrene (PX40) fibers, in a hollow fiber bioreactor for the attachment and proliferation of skeletal muscle cells. For the immortalized mouse myoblast cell line, C2C12s, cells were confirmed to be alive with approximately 80% viability after 5 days of culture based on trypan blue assessment.

[0184] Example 3 Cell Adhesion Assay (Protocol Overview) Desired cell count (cell suspension concentration per mL and corresponding volume in mL to reach target seeding density). A suspension volume equal to or less than the ECS volume was initially injected into the reactor, then replenished with fresh medium to avoid cell loss if not all of the ECS volume from the original seeding suspension volume was injected into the reactor's side port.

[0185] The module was attached to a specially designed saddle clip on the rotor at a specific angle (9.2 degrees in this case).

[0186] Constant rotation was applied to ensure proper distribution of cells throughout the fiber bundle.

[0187] The module was removed, the growth medium was drained through a side port to remove unattached cells, replaced with fresh growth medium, and connected to a continuous dynamic delivery system after seeding.

[0188] The seeding method consisted of the perfusion module being tilted at 9.2 degrees and continuously rotating at 0.3 RPM, with the module offset from the axis of rotation by 150 mm.

[0189] Cell viability was determined by either direct or indirect measurements of cell viability.

[0190] Direct measurement - Cells were seeded at the required density and allowed to adhere for a predetermined period. Cells were harvested from excised fibers and quantified by direct cell counting (Chemometec NC-200) to calculate VCC (viable cell count).

[0191] Indirect measurement - Cells were seeded at the required density and allowed to attach for a predetermined period of time. The module was drained to exchange the growth medium. The cells contained in the drained growth medium were quantified as total cell number (including non-viable cells). Indirect attachment efficiency = (cells seeded - cells removed during draining after seeding) / cells seeded (%).

[0192] (result) Increasing the time improves the initial attachment strength and therefore the attachment efficiency when quantified directly* (see "Test Objective - Attachment Efficiency" experiment) or indirectly** (all other experiments). Cells gradually form an ECM layer, which means that they are initially more loosely attached and therefore the attachment strength improves.

[0193] (Consideration) The data set shows that for attachment efficiency experiments, values ​​range from 35.4% to 71.4%. These measurements are obtained by directly measuring the cells removed using a cell removal agent, which is then quantified by direct cell counting (Chemometec NC-200) to obtain the VCC (viable cell count). [Table 2] JPEG2025528869000004.jpg229145JPEG2025528869000005.jpg229152

[0194] The reader is directed to all articles and documents related to this application that have been filed contemporaneously or previously hereto and are published herewith, the contents of all such articles and documents being incorporated herein by reference.

[0195] All features disclosed in this specification (including any accompanying claims, abstract, and drawings), and / or all steps of any disclosed method or process, may be combined in any combination, except combinations in which at least some of the features and / or steps are mutually exclusive.

[0196] Each feature disclosed in this specification (including the accompanying claims, abstract, and drawings), unless otherwise stated, may be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless otherwise stated, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0197] The invention is not limited to the details of the foregoing embodiments, and extends to any novel one or any novel combination of features disclosed in this specification (including the accompanying claims, abstract and drawings), or any novel one or any novel combination of steps of the disclosed methods or processes.

[0198] <References>

[0199] [1] K.A. Luetchford et al., "Next generation in vitro liver model design: Combining a permeable polystyrene membrane with a transdifferentiated cell line", J. Memb. Sci., vol. 565, pp. 425 - 438, Nov. 2018. [2] M.P. Storm et al., "Hollow Fiber Bioreactors for In Vivo - like Mammalian Tissue Culture", J. Vis. Exp., no. 111, pp. 1 - 12, 2016. [3] S.M. Acott, "Cell Expansion And Delivery Methods For Heart Regeneration[Thesis]", University of Bath, 2016. [4] Mulder, M. The basic principles of membrane technology. 2nd ed. Chapter 3 section 4. Kluwer Academic Publishers(1996). [5] Ellis, M.J., Chaudhuri, J.B. Poly(lactic - co - glycolic acid) hollow fibre membranes for use as a tissue engineering scaffold. Biotechnol Bioeng. 96(1), 177 - 187(2007). [6]Shearer,H.、Ellis,M.J.、Perera,S.P.、Chaudhuri,J.B.Effects of common sterilization methods on the structure and properties of poly(D,L lactic-co-glycolic acid)scaffolds.Tissue Eng.12(10)、2717-2727(2006)。

Claims

1. 1. A method for culturing muscle cells for an edible product, comprising: a) providing a perfusion module for a perfusion bioreactor system, the perfusion module comprising one or more porous hollow fibers comprising an exterior surface and an interior lumen; b) seeding muscle cells onto the one or more porous hollow fibers; c) maintaining the perfusion module with the one or more porous hollow fibers oriented at an angle of between 0 and 90 degrees from the horizontal for a period of time sufficient for the muscle cells to initially adhere to the outer surface of each of the one or more porous hollow fibers; d) rotating the perfusion module; e) connecting the perfusion module to a perfusion bioreactor system; and f) culturing the muscle cells attached to the exterior surface of each of the one or more porous hollow fibers under conditions suitable for proliferation and / or differentiation of the muscle cells. The method comprising:

2. The method of claim 1 , wherein the period of time is at least 10 minutes.

3. 3. The method of claim 1 or 2, wherein the angle is between 0 and 30 degrees from horizontal; preferably between 5 and 20 degrees; and more preferably between 8 and 10 degrees.

4. The method of any one of claims 1 to 3, wherein the rotating comprises applying a centrifugal force of 0 to 50N.

5. The method of any one of claims 1 to 4, wherein rotating comprises rotating continuously; optionally rotating continuously at a speed of 0 to 30 rpm.

6. The method of any one of claims 1 to 4, wherein rotating the perfusion module comprises intermittently rotating the perfusion module.

7. 7. The method of claim 6, wherein rotating the perfusion module comprises rotating at a speed of 0-30 rpm for about 1 second to about 5 minutes every 10 seconds to about 30 minutes for a total time of at least 1 hour; and optionally rotating at a speed of about 12 rpm for about 30 seconds every about 5 minutes.

8. 10. The method of any preceding claim, wherein rotating the perfusion module comprises rotating for at least 3 hours; optionally, rotating for about 3-5 hours.

9. 10. The method of any preceding claim, wherein rotating the perfusion module comprises rotating the perfusion module with an offset between a midpoint of a centerline of the perfusion module and an axis of rotation; optionally, the offset is at least 0.01 mm.

10. 10. The method of any preceding claim, wherein the attachment efficiency is at least 55% as quantitatively determined by direct or indirect measurement of viable cells attached to the hollow fibers.

11. Prior to seeding, the muscle cells: (i) washing with a composition comprising a cell detachment agent; (ii) harvested in a culture medium containing a growth promoter; (iii) centrifuging to form a pellet; (iv) resuspending in culture medium to form a cell suspension; 10. A method according to any preceding claim.

12. 12. The method of claim 11, wherein seeding the muscle cells comprises applying the cell suspension containing the muscle cells to the perfusion module.

13. 10. The method of any of the preceding claims, wherein seeding the muscle cells comprises applying a cell density about 0.1 to 10 times higher than used on 2D tissue culture plastic.

14. Seeding the myocytes in the perfusion module may include seeding the myocytes in the perfusion module at least 100 cells / cm. ２ 10. The method of any preceding claim, comprising applying

15. the one or more porous hollow fibers It is hydrophilic; washable; and / or It is reusable, 10. A method according to any preceding claim.

16. 10. The method of any preceding claim, wherein the one or more porous hollow fibers comprise polystyrene.

17. The method of any one of claims 1 to 15, wherein the one or more porous hollow fibers are biodegradable and / or edible.

18. culturing includes pumping a culture medium through the perfusion bioreactor system and the interior lumen of each of the one or more porous hollow fibers; Optionally, culturing further comprises maintaining the perfusion module in an orientation such that the one or more porous hollow fibers are at an angle of 0 to 90 degrees from horizontal; optionally, the angle is 0 to 30 degrees, preferably 5 to 20 degrees, more preferably 8 to 10 degrees from horizontal.

10. A method according to any preceding claim.

19. 10. The method of any preceding claim, wherein culturing comprises pumping culture medium through the perfusion bioreactor system and the interior lumen of each of the one or more porous hollow fibers at a rate of at least 10 μL / hr / hollow fiber.

20. Culturing the muscle cells at a temperature of at least 15° C. and / or 5% CO ２ 10. The method of any preceding claim, comprising maintaining

21. 10. The method of any of the preceding claims, wherein culturing comprises pumping culture medium through the perfusion bioreactor system and perfusion module for at least 3 hours.

22. 10. The method of any preceding claim, wherein culturing comprises pumping a first culture medium through the perfusion bioreactor system and the interior lumen of each of the one or more porous hollow fibers, and optionally, the first culture medium is a growth medium.

23. 23. The method of claim 22, wherein culturing further comprises pumping a second culture medium through the perfusion bioreactor system and the interior lumen of each of the one or more porous hollow fibers, optionally wherein the second culture medium is a differentiation medium; and further optionally, wherein the differentiation medium is perfused through the bioreactor system and perfusion module for at least 3 hours.

24. 10. The method according to any of the preceding claims, wherein the perfusion bioreactor system comprises a device for monitoring metabolite and / or oxygen concentrations in the culture medium.

25. 10. The method of any preceding claim, further comprising: (g) harvesting the muscle cells from the perfusion module.

26. forming the harvested muscle cells into a cultured meat product; or the one or more hollow fibers are edible, and the harvested cells and the one or more porous hollow fibers are formed into a cultured meat product.

26. The method of claim 25.

27. 10. The method of any preceding claim, wherein the muscle cells are derived from at least one food animal cell; optionally, the muscle cells comprise one or more of fibroblasts, skeletal muscle cells, smooth muscle cells, and / or myoblasts; further, optionally, the muscle cells are derived from one or more of non-human embryonic stem cells and / or pluripotent stem cells.

28. An edible product comprising muscle cells obtained by the method of any one of claims 1 to 27.

29. 28. An edible product obtainable by the method of any one of claims 26 and 27.

30. 30. The method of any one of claims 1 to 27, or the edible product of claims 28 or 29, wherein the edible product is a cultured meat product.

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