WO2024038281A1 - Procédé de culture de cellules musculaires avec bioréacteur à perfusion - Google Patents

Procédé de culture de cellules musculaires avec bioréacteur à perfusion Download PDF

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Publication number
WO2024038281A1
WO2024038281A1 PCT/GB2023/052165 GB2023052165W WO2024038281A1 WO 2024038281 A1 WO2024038281 A1 WO 2024038281A1 GB 2023052165 W GB2023052165 W GB 2023052165W WO 2024038281 A1 WO2024038281 A1 WO 2024038281A1
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cells
perfusion
hollow fibres
cell
module
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PCT/GB2023/052165
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English (en)
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Professor Marianne Jane ELLIS
Illtud Llyr DUNSFORD
Scott James ALLAN
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Cellular Agriculture Ltd
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Publication of WO2024038281A1 publication Critical patent/WO2024038281A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/10—Hollow fibers or tubes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/02—Hollow fibre modules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/16—Hollow fibers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
    • C12M3/062—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means with flat plate filter elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels

Definitions

  • the present invention provides methods of culturing muscle cells.
  • methods of culturing muscles cells using a hollow fibre bioreactor HFB.
  • comestible products including or formed from the cells produced from by the methods or the cells and hollow fibres used in the methods.
  • bioreactors with higher cell densities allow a smaller culture volume thus reducing space requirements, labour requirements to set up and harvest the cells, and the amount of raw materials to manufacture them. Operating costs will be also lower as smaller bioreactors requiring less power and utilities.
  • Native skeletal muscle anatomy consists of several arrays of uniaxial, striated myofibres in conjunction with fat cells, fibroblasts, capillaries and veins.
  • a capillary is connected to most of the myofibres in a fascicle to provide blood perfusion, to provide the muscle cells with adequate oxygen and nutrients and also take away cell metabolism waste.
  • This structure is well-replicated in a hollow fibre bioreactor (HFB) where the inlet media carries oxygen and nutrients, goes through the fibres and nourishes the cells and the perfused flow (permeate) and/or retentate carries out the wastes at an outlet.
  • HFB hollow fibre bioreactor
  • Hollow fibre bioreactors are classified as a hydraulic bioreactor, meaning mixing is achieved via liquid flow rather than by mechanical mixing. This involves seeding the cells in a matrix with porous hollow fibres to allow cells to adhere to the hollow fibre surface where the medium may also circulate.
  • a hollow fibre system offers the benefits of creating low shear stress, increased selection of which nutrients are transported, and is ideal for highly metabolic cell types.
  • hollow fibre bioreactors are mostly limited to cell culture and do not support culturing tissue scaffolds.
  • the invention is based on the surprising finding that the use of the methods described herein provide high yields of muscle cells suitable for use in or production of meat analogues. For example, it has been found that maintaining the prefusion module at an angle of between about 0 to 90 degrees for a period of time allows for attachment of the cells to the hollow fibres. It has also been found that culturing cells at an angle of between about 0 to 90 degrees may allow for higher amounts of cells to be produced.
  • a method of culturing muscles cells for a comestible product comprising: a) providing a perfusion module for a perfusion bioreactor system; the perfusion module comprising one or more porous hollow fibres comprising and outer surface and an internal lumen; b) seeding muscle cells onto the one or more porous hollow fibres; c) maintaining the perfusion module in an orientation wherein the one or more porous hollow fibres is at an angle between 0 to 90 from horizontal for a time period sufficient to allow initial attachment of the muscle cells to an outer surface of each of the one or more porous hollow fibres; 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 fibres under conditions suitable for proliferation and/or differentiation of the muscle cells.
  • the time period is at least 10 minutes.
  • the angle is between 0 and 30 degrees from horizontal. In certain embodiments, the angle is between 5 and 20 degrees from horizontal. In certain embodiments, the angle is between 8 and 10 degrees from horizontal. In some embodiments, the angle is about 9.2 degrees.
  • rotating comprises exerting a centrifugal force of between 0 and 50 N.
  • a centrifugal force of between 0 and 50 N.
  • rotating comprises continuously rotating. In certain embodiments, rotating comprises continuously rotating at a speed of between 0 and 30 rpm. In certain embodiments, rotating comprises continuously rotating at a speed of about 1 to 4 rpm. In certain embodiments, rotating comprises continuously rotating for at least 1 hour. In certain embodiments, rotating comprises continuously rotating for at least 3 hour. In certain embodiments, rotating comprises continuously rotating for about 3 to 5 hours.
  • rotating the perfusion module comprises rotating the perfusion module with an offset between the mid-point of the centreline of the perfusion module and the axis of rotation.
  • the offset is greater than 0mm.
  • the offset is at least 10 mm.
  • the offset is up to 500mm. In certain embodiments, the offset is about 150 mm.
  • the attachment efficiency is at least 5% as measured by quantification of viable cells attached to the hollow fibres by direct or indirect measurement. In certain embodiments, the attachment efficiency is at least 10%, 20%, 30%, 40% or 50% as measured by quantification of viable cells attached to the hollow fibres by direct or indirect measurement In certain embodiments, the attachment efficiency is at least 55% as measured by quantification of viable cells attached to the hollow fibres by direct or indirect measurement.
  • rotating the perfusion module comprises intermittently rotating the perfusion module. In certain embodiments, rotating the perfusion module comprises rotating at a speed of between 0 and 30 rpm for about 1 second to about 5 minutes about every 10 seconds to about 30 minutes for a total time of at least 1 hour.
  • rotating the perfusion module comprises rotating at a speed of about 12 rpm for about 30 seconds about every 5 minutes.
  • rotating the perfusion module comprises rotating for at least three hours.
  • the muscle cells prior to seeding the muscle cells are:
  • seeding the muscle cells comprises applying the cell suspension comprising the muscle cells to the perfusion module.
  • seeding the muscle cells comprises applying around at least 0.1 -fold higher cell density than used for 2D tissue culture plastic. For example, 0.1 to 10 fold higher cell density.
  • seeding the muscles cells comprises applying at least 1000 cells/cm 2 to the perfusion module.
  • the one or more porous hollow fibres are hydrophilic; are washable; and/or are reusable.
  • the one or more porous hollow fibres comprise polystyrene.
  • the one or more porous hollow fibres are biodegradable and/or edible.
  • culturing comprises pumping culture media through the perfusion bioreactor system and the internal lumen of each of the one or more porous hollow fibres; optionally wherein culturing further comprises maintaining the perfusion module is maintained in an orientation wherein the one or more porous hollow fibres is at an angle between 0 to 90 from horizontal.
  • the angle is between 0 and 30 degrees from horizontal; preferably between 5 and 20 degrees; more preferably between 8 and 10 degrees
  • culturing comprises pumping culture media through the perfusion bioreactor system and the internal lumen of each of the one or more porous hollow fibres at a rate of at least 10pL/hour/hollow fibre.
  • culturing comprises maintaining the muscle cells at a temperature of at least 15°C and/or 5% CO2.
  • culturing comprises pumping culture media through the perfusion bioreactor system and perfusion module for at least 3 hours.
  • culturing comprises pumping a first culture media through the perfusion bioreactor system and the internal lumen of each of the one or more porous hollow fibres, optionally wherein the first culture media is a proliferation medium.
  • culturing further comprises pumping a second culture media through the perfusion bioreactor system and the internal lumen of each of the one or more porous hollow fibres, optionally wherein the second culture media is a differentiation medium; further optionally wherein the differentiation media is perfused through the bioreactor system and perfusion module for at least 3 hours.
  • the perfusion bioreactor system comprises apparatus for monitoring metabolite concentration and/or oxygen concentration of the culture media.
  • the method further comprises:
  • the harvested muscle cells are formed into a cultured meat product; or wherein the one or more hollow fibres are edible, and the harvested cells and the one or more porous hollow fibres are formed into a cultured meat product.
  • the muscle cells are derived from at least one comestible animal cell; optionally wherein the muscle cells comprise one or more of fibroblasts, skeletal muscle cells, smooth muscle cells, and/or myoblasts; further optionally wherein the muscle cells ae derived from one or more of non-human embryonic stem cells and/or pluripotent stem cells.
  • a comestible product comprising muscle cells obtained by a method as described herein.
  • the comestible product is a cultured meat product.
  • Figure 1 shows the in vivo-like environment of a HFB.
  • Cells are seeded onto the outside of the porous fibres. Media is delivered through the fibre lumen, mimicking a blood capillary.
  • A Longitudinal section of a fibre (not to scale).
  • B Cross section of a 3 fibre reactor;
  • Figure 2 shows an HFB module.
  • A The dimensions of the module. The dimensions were chosen to fit 3 fibres. Different sizes can be manufactured and tailored for individual systems and fibres.
  • B A photograph of the module with attached end cap and module connectors.
  • FIG. 3 shows module fabrication.
  • A Fibres are cut to size and inserted into the module.
  • B Fibres are glued into the module, allowed to dry.
  • C The ends are cut flush with the glass.
  • FIG. 4 shows HFB system setup. Arrows indicate direction of media flow.
  • A feed tube.
  • B pump tube.
  • E HFB module.
  • D retentate tube with clamp.
  • C permeate tube.
  • Figure 6 shows relationship between midpoint of the (axial) centreline of a perfusion module to axis of rotation in determining offset.
  • Perfusion culturing refers to a continuous culturing method in which cells are either retained in the bioreactor or fed back into it.
  • the cell culture medium perfused through the bioreactor thus contains no cells.
  • Perfusion-based culturing methods may result in higher cell concentrations and product yields in the reactor while reducing the working volume, for example, in view of continuous stirred tank reactors methods and systems.
  • the term “perfusion bioreactor” refers to a cell culture system in which the cell culture medium (e.g., a first cell culture medium, a second cell culture medium, a culture medium, a cell proliferation cell culture medium, and/or a cell differentiation cell culture medium) is continuously replaced with fresh media.
  • Perfusion bioreactor systems may include means (e.g., an outlet, inlet, pump, or other such devices) for periodically or continuously withdrawing and adding substantially the same volume of replacement cell culture medium to the bioreactor.
  • the addition of the replacement liquid culture can be performed substantially simultaneously with or immediately after the removal of the initial cell culture medium from the bioreactor.
  • the means for removing the liquid culture from the bioreactor and for adding the replacement liquid culture may be a single device or system.
  • the means for removing and replacing i.e. perfusing cell culture media
  • a “bioreactor” may be any device or system that maintains a biologically active environment, for example, a chamber or vessel in which cells can be cultured.
  • a biologically active environment for example, a chamber or vessel in which cells can be cultured.
  • bioreactor types that differ in shape (e.g., cylindrical or otherwise), size (e.g., millilitres, litters to cubic meters), and materials (stainless steel, glass, plastic, etc.). Accordingly, the bioreactor is adapted to grow cells or tissue in cell culture.
  • the bioreactor may be configured to receive hollow fibres as described herein.
  • a hollow fibre perfusion bioreactor includes a perfusion module (which may also be referred to as a bioreactor) with hollow fibres contained therein.
  • a hollow fibre perfusion bioreactor system as used in the methods described herein includes a continuous perfusion system with a constant replenishment of nutrients and removal of waste.
  • Medium circulates through a perfusion module, including porous hollow fibres.
  • the interior of the hollow fibres may be termed the Intracapillary space (ICS) or internal lumen, and the exterior may be termed the extracapillary space (ECS).
  • ICS Intracapillary space
  • ECS extracapillary space
  • Cells grow in the ECS and adhere to the outside of the fibres (outer surface).
  • the fibres may provide a large surface area for cell contact in a relatively compact system.
  • a schematic diagram of the general concept of a hollow fibre system is shown in Figure 1. As can be seen in Figure 1 , media (1) flows through the lumen (10) of a hollow fibre (12). Pores (14) in the hollow fibre allow for the transfer of nutrients to cells (16) which adhere to the outer surface (18) of the hollow fibre.
  • small molecular weight cellular nutrients and metabolic waste products can pass between the ICS and the ECS via the pores of the porous hollow fibres, whilst cells may be confined to the ECS.
  • One or more “side” ports may be used to introduce large molecular weight nutrients and/or flow media or liquids into the ECS to provide defined shear rates to the cells.
  • An additional side port may be used to collect fluid flowing through the ECS.
  • HFBs offer an in vivo-like environment with the fibres mimicking blood capillaries and shielding the cells from the shear stresses associated with dynamic media delivery while allowing defined shear to be applied to cells via fluid flow through the side ports if desired. This creates a versatile culture system with superior mass transport in which high cell densities can be reached.
  • FIG. 2A An example of a perfusion module is shown in Figures 2 and 3.
  • FIG 2A a schematic of an example perfusion module is shown.
  • the perfusion module (2) includes an inlet port (20) and an outlet port (21) located at each end 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 hollow fibres (3) inserted into the perfusion module channel (32).
  • FIG. 4 An example perfusion system setup is shown in Figure 4.
  • the system includes a pump system (4) connected to a perfusion module (40 and E).
  • Culture media is fed from a reservoir (42) via a feed tube (A) to the pump system (4), which then pumps media to the perfusion module via pump tubing (B).
  • the culture media flows through the perfusion module and exits from the lumens of the hollow fibres via retentate tubing (D) and media that has exchanged through the pores of the hollow fibres and into ECS via permeate tubing (C).
  • the pumping system may be any suitable pump.
  • the pump system may be a peristaltic pump system or vacuum pump system.
  • the flow rate of culture media that is flowed through the perfusion module may be controlled by the pump system.
  • the flow rate may be selected depending on the number of hollow fibres being used, the cells being cultured and/or the number of cells seeded onto the outer surface of the hollow fibres.
  • culture media may be pumped at a rate of at leastl pL/hour/hollow fibre. That is to say, for each hollow fibre used, the flow rate is 1 pL/hour. In some examples, the flow rate is at least 10 pL/hour/hollow fibre.
  • the flow rate is from 100 to 300 pL/hour/hollow fibre. In some examples, the flow rate is between 240 to 280 pL/hour/hollow fibre. In some examples, the flow rate is about 267 pL/hour/hollow fibre.
  • the perfusion bioreactor system may further include monitoring apparatus.
  • Monitoring apparatus refers to any apparatus suitable for determining and/or monitoring changes in one or more properties of cells being cultured and/or one or more properties of the perfused media after having flowed through the hollow fibres (retentate) and/or that has passed though the pores (permeate).
  • the monitoring apparatus may be a system for monitoring nutrient and/or metabolite content of the permeate and/or retentate.
  • permeate refers to culture media that has passed through the pores of the hollow fibres (i.e. permeated through the hollow fibres).
  • retentate refers to culture media that flows through the internal lumen of the hollow fibres (i.e. is retained within the hollow fibres).
  • Nutrients that may be monitored may be any nutrient and/or metabolite that is included in the culture media used.
  • the nutrient and/or metabolite may be one or more of a saccharide (such as 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 or the like.
  • the concentrations of nutrients and/or metabolites may be compared to the initial concentration of the corresponding nutrients and/or metabolites in the culture media prior to culturing the cells or prior to being perfused through the hollow fibres. Changes in concentrations of nutrients and/or metabolites in the permeate and/or retentate in comparison to the initial cell culture media may provide information on the rate of proliferation, cell number and/or condition (e.g. viability and/or health) of the cells being cultured.
  • systems for monitoring concentrations of nutrients and/or metabolites may be separate from the perfusion bioreactor system.
  • the methods may further include collecting a portion of the permeate and/or retentate at predetermined time points and determining the concentrations of nutrients and/or metabolites.
  • the perfusion bioreactor system may include a system for monitoring nutrients and/or metabolites that is connected directly to the perfusion bioreactor system.
  • Suitable methods for determining concentrations of nutrients and/or metabolites in culture media are well known in the field. For example, kits such as those provided by Megazyme are available that include instructions therein.
  • the monitoring apparatus may include a system for monitoring a concentration of dissolved oxygen in the permeate and/or retentate.
  • concentrations of dissolved oxygen may be compared to the initial concentration of dissolved oxygen in the culture media prior to culturing the cells or prior to being perfused through the hollow fibres.
  • Changes in concentrations of dissolved oxygen in the permeate and/or retentate in comparison to the initial cell culture media may provide information on the rate of proliferation, cell number and/or condition (e.g. viability and/or health) of the cells being cultured.
  • Systems for determining the concentration of dissolved oxygen in culture media include, for example, PreSens flow-through cell and PreSens Fibox 4 oxygen reader.
  • the cells seeded onto the hollow fibres include muscle cells.
  • Muscle cells include those cells making up 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 and change the size of the cell. They are classified as skeletal, cardiac, or smooth muscles.
  • the term “cells that can differentiate into muscle cells” refers to stem cells and muscle progenitor cells that can differentiate into muscle cells (e.g. mature muscle cells).
  • Muscle cells may include those cells normally found in muscle tissue, including smooth muscle cells, cardiac muscle cells, skeletal muscle cells (e.g., muscle fibres or myocytes, myoblasts, myotubes, etc.), and any combination thereof. Muscle cells may include myoblasts, myotubes, myofibrils, and/or satellite cells.
  • the cells may further include adipose or fat cells.
  • Adipose or fat cells include any cell or group of cells composed in a fat tissue, including, for example, lipocytes, adipocytes, adipocyte precursors including, pre-adipocytes and mesenchymal stem cells.
  • the cells may be derived from any source animal. As the perfusion modules described herein may be for use in making comestible products, the cells may not be derived from a human.
  • the cells may be derived from bovine, ovine, equine, porcine, caprine, avian, fish, insect, crustaceans, cephalopod, mollusc and/or camelid animals.
  • the cells may be derived from a bovine, porcine, avian and/or ovine animal.
  • the cells may be derived from a cow, pig, chicken, fish, squid, insect, oyster and/or sheep.
  • the cell culture medium that may be used in the methods described herein may be any suitable cell culture medium.
  • the cell culture medium may be selected depending on the type of cell cells being cultured. Examples of culture mediums that may be used include minimal essential medium (MEM, Sigma, St. Louis, Mo); Dulbecco’s modified Eagle medium (DMEM, Sigma); Ham F10 medium (Sigma); Cell culture media (HyClone, Logan, Utah); RPMI-1640 culture media (Sigma); and chemical-defined (CD) culture media (which are formulated for individual cell types), such as CD-CHO culture media (Invitrogen, Carlsbad, Calif).
  • the culture solution described above can be supplemented with auxiliary components or contents as needed. This includes any component of the appropriate concentration or amount required or desired.
  • the culture medium described above can be supplemented with auxiliary components or contents as needed.
  • the culture medium may include one or more additives such as antibiotics, proteins, amino acids and/or sugars.
  • “Medium” and “cell culture medium” refer to a nutrient source used for growing or maintaining cells. As is understood by a person of skill in the art, the nutrient source may contain components required by the cell for growth and/or survival or may contain components that aid in cell growth and/or survival. Vitamins, essential or non-essential amino acids, trace elements, and surfactants (e.g., poloxamers) are examples of medium components. Any media provided herein may also be supplemented with any one or more of insulin, plant hydrolysates and animal hydrolysates.
  • “Culturing” a cell refers to contacting a cell with a cell culture medium under conditions suitable for the viability and/or growth and/or proliferation of the cell.
  • Perfusing the cell culture medium may include perfusing a first culture media and then subsequently perfusing one or more second cell culture mediums.
  • the first cell culture medium may be a cell culture medium that is for proliferating cells and may be referred to as proliferation medium.
  • Proliferation medium may be a medium comprising a source of nutrients, such as vitamins, minerals, carbon and energy sources, and other beneficial compounds that facilitate the biochemical and physiological processes occurring during expansion or proliferation of cells.
  • the proliferation medium may comprise one or more carbon sources, vitamins, amino acids, and inorganic nutrients.
  • Representative carbon sources include monosaccharides, disaccharides, and/or starches.
  • the proliferation medium may contain one or more carbohydrates such as sucrose, fructose, maltose, galactose, mannose, and lactose.
  • the proliferation medium may also comprise amino acids.
  • Suitable amino acids may include amino acids commonly found incorporated into proteins as well as amino acids not commonly found incorporated into proteins, such as arginosuccinate, citrulline, canavanine, ornithine, and D-stereoisomers.
  • the proliferation medium may also comprise growth promotion agents, such as serum, for example, foetal bovine serum (FBS).
  • growth promotion agents include growth factors (e.g. recombinant growth factors), bovine ocular fluid, sericin protein, earthworm heat inactivated coelomic fluid.
  • the proliferation media may be a serum free culture media and may optionally include additional components depending on the cells being cultured.
  • serum free culture medias include those commercially available from ThermoFisher, Lonza Bioscience and Merck.
  • the proliferation medium may also comprise antibiotics.
  • the proliferation medium may be Dulbecco’s Modified Eagle’s Medium (DMEM), which may include 10% (V/V) filter sterilised foetal bovine serum and 1% (V/V) penicillin/streptomycin solution.
  • DMEM Dulbecco’s Modified Eagle’s Medium
  • the proliferation media may be a media such as Gibco insect media available from ThermoFisher.
  • the cells may be maintained and cultured in proliferation 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.
  • the cells may be maintained in proliferation media continuously.
  • the cell culture media may be changed to a second cell culture medium.
  • the second cell culture medium may be a differentiation medium.
  • Differentiation medium refers to a medium designed to support the differentiation of cells, that is, supporting the process of a cell changing 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 include one or more proteins, amino acids or other additional acids.
  • the differentiation media includes one or more growth promotion agents, such as serum, for example, foetal bovine serum or horse serum. Examples of other growth promotion agents include growth factors (e.g.
  • the differentiation media may be a serum free culture media and may optionally include additional components depending on the cells being cultured.
  • serum free culture medias include those commercially available from ThermoFisher, Lonza Bioscience and Merck.
  • the differentiation medium may be high-glucose DMEM (97%) supplemented with 2% horse serum and 1% penicillin/streptomycin solution.
  • the cells may be maintained and cultured in 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.
  • the culture media may be changed a number of times.
  • the cells may first be cultured in proliferation media; then, the culture media is switched to differentiation media.
  • the differentiation media may then be switched to proliferation media.
  • the proliferation media may be the same as before or may contain different components and/or concentrations of components to the initial proliferation media.
  • the cells may be continuously cultured. That is to say that the cells are maintained in one or more culture mediums as described herein.
  • the cells may be cultured for a first time period (e.g. at least 3 hours or more) in a first media, then for a second time period (e.g. at least 3 hours or more) in a second media and then cultured in a further media and continuously cultured with any number of changes of the culture media so that cells can be constantly proliferated and/or differentiated.
  • the cells may be harvested at predetermined cell densities or time points in order to avoid overcrowding, reduced viability and/or cell death.
  • the cell culturing conditions may be selected depending on the cell type and/or cell source.
  • cells may be cultured (e.g. proliferated and/or differentiated) at a temperature of at least 15°C.
  • the cells may 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.
  • the cells may be cultured at a temperature of from 15°C to 32 °C.
  • the cells may be cultured at a temperature of 37°C.
  • cells may be cultured at a temperature of from 15°C to 32 °C. In some examples, marine animal cells may be cultured at a temperature between 15°C to 30 °C.
  • the cells may be cultured in defined atmospheric conditions.
  • the cells may be cultured in an atmosphere that has predetermined humidity and/or gas concentration.
  • cells may be cultured in an atmosphere including at least 5% CO 2 .
  • Polymers that may be used to form the hollow fibres may be any polymer suitable for culturing and/or maintenance of cells. Suitable polymers include biodegradable polymers.
  • the polymer may be a biocompatible polymer.
  • Biodegradable polymers are any polymers that may be broken down by biological systems, such as polymers that can be broken down into harmless products by the action of living organisms. Biocompatible polymers are, along with any metabolites or degradation products thereof, generally nontoxic to cells or to a recipient (such as a human or animal) and do not cause any significant adverse effects to cells or a recipient at concentrations resulting from the degradation of the polymers.
  • biocompatible polymers are polymers that do not elicit result in negative effects on cell health or in a recipient.
  • biocompatible and/or biodegradable polymers may be advantageous if the fibres or part thereof is consumed (for example, ingested) by a person.
  • Biodegradable polymers include linear aliphatic polyesters such as polylactic acid, polyglycolic acid, polycaprolactone, polyhydroxybutyrate, polyhydroxyvalerate and their copolymers within the aliphatic polyester family such as poly(lactic-co-glycolic acid) and poly(glycolic acid-co-caprolactone); copolymers of linear aliphatic polyesters and other polymers such as poly(glycolic acid-co-trimethylene carbonate) copolymers, poly(lactic acid- co-lysine) copolymers, tyrosine-based polyarylates or polyiminocarbonates or polycarbonates, poly(lactide-urethane) and poly(ester-amide) polymers; polyanhydrides such as poly(sebacic anhydride); polyorthoesters such as 3,9-diethyidiene-2,4,8,10-tetraoxaspiro- 5,5-undecane based polymers; poly(ester-amide
  • the polymers may be edible polymers.
  • Edible polymers refers to any polymer that is acceptable for use in an edible product.
  • Examples of edible polymers include polyvinyl alcohol, carboxyvinyl polymer, hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose, ethylcellulose, low- substituted hydroxypropylcellulose, crystalline cellulose, carboxymethylcellulose sodium, a synthetic polymer compound such as carboxymethylcellulose calcium, carboxymethylcellulose and carboxymethylstarch sodium, sodium alginate, dextran, casein, pullulan, pectin, guar gum, xanthan gum, tragacanth gum, acacia gum, zein, gelatin, chitin and chitosan, silk, fibrin and polymer compounds obtained from natural products such as starch or soybean.
  • an edible polymer may provide hollow fibres and products, including the hollow fibres which are edible.
  • a cell culture grown on the hollow fibres may provide for an edible product that does not require the removal of the hollow fibres before consumption.
  • the hollow fibres may be a digestible polymer.
  • “Digestible” refers to a material that, when eaten by a subject, can be broken down into compounds that can be absorbed and used as nutrients or eliminated by the subject’s body.
  • Digestible polymers include BCS, polylactic acids (PLAs), synthetic polyamides, polycarbonates, polyisocyanurates (PIRs), polyurethanes, polyethers, proteins, polysaccharides (such as starches), polylactones, polylactams or glycols.
  • the hollow fibres include poly(lactic-co-glycolic acid) (PLGA).
  • PLGA poly(lactic-co-glycolic acid)
  • the hollow fibres may include 10% PLGA.
  • the hollow fibres include a reusable polymer.
  • a reusable polymer refers to a polymer that does not degrade over time or due to use in culturing cells. Reusable polymers may provide a perfusion module that can be washed and used multiple times.
  • the hollow fibres are hydrophobic.
  • the hollow fibres are washable. Washable refers to hollow fibres that can be washed and/or sterilised without sustaining damage or loss of function. Hollow fibres with such properties may reduce waste and costs.
  • the hollow fibres may be made from polystyrene.
  • Hollow fibres may be solid or semi-solid substrates having openings or apertures (pores), which allow components of cell culture media, such as metabolites, nutrients and gases (e.g. oxygen), to be delivered to cells attached to the outer surfaces of the hollow fibres.
  • Hollow fibres described herein allow the growth of cells on the outer surface of the hollow fibres.
  • the hollow fibres described herein act as a 3-dimensional matrix that allows for the culture and maintenance of cells in a 3-dimensional architecture.
  • the hollow fibres may have a Young’s modulus of at least 1000 Pa. In some examples, the hollow fibres have a Young’s modulus from 1000 Pa to 1000000000 Pa. In some examples, the hollow fibres have a Young’s modulus between 8000 Pa to about 20000 Pa.
  • the hollow fibres may have a Young’s modulus of around 115 MPa.
  • the pores of the porous polymer sheets may have an average pore diameter suitable for allowing infiltration and support cells within the polymer sheet.
  • the pores may have an average diameter from at least 0.001 pm to 100 pm.
  • the porous scaffold may have pores with an average pore diameter of around 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, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 pm.
  • the pores may have an average diameter from 0.001 pm to 5 pm.
  • the pores may have an average diameter of around 2.5 pm. In some examples, the pores may have an average pore diameter of around 5 pm.
  • Average pore size may be determined using optical methods such as scanning electron microscopy (SEM), atomic force microscopy (AFM), computed tomography methods and/or transmission electron microscopy (TEM). Other methods that may be used include X- ray refraction methods, imbibition methods, mercury injection methods, and gas expansion methods.
  • SEM scanning electron microscopy
  • AFM atomic force microscopy
  • TEM transmission electron microscopy
  • Other methods that may be used include X- ray refraction methods, imbibition methods, mercury injection methods, and gas expansion methods.
  • the average pore size and porosity may affect the penetration of cells into the scaffold and define the spatial distribution of cells within the 3D matrix of the scaffold.
  • average pore size and porosity may affect the flow resistance, the transportation of nutrients, and/or the excretion of waste products from cells cultured thereon and/or therein.
  • the lumens of each hollow fibre act as conduits to transport cell culture medium to the cells located on the outer surface of the hollow fibres, as well as transporting waste products from the cells.
  • the density of pores on each hollow fibre may be at least 1 pore/mm 2 .
  • the pore density may be around 210 pores/mm 2 .
  • the lumens of each hollow fibre may have an internal diameter from 1 pm to 1000pm.
  • the internal diameter refers to the diameter measured between the inner surface of the lumen.
  • the inner lumen diameter may be about 500pm.
  • the inner lumen diameter is from about 600 to about 700 pm. In some examples, the inner lumen diameter may be around 630 to 660 pm. In some examples, the inner lumen diameter is from about 50 to about 200 pm.
  • Each of the hollow fibres may have an outer diameter of between about 12 pm and 2000 pm.
  • each of the hollow fibres may have an outer diameter from 500 to about 1000 pm.
  • the outer diameter of each hollow fibre may have an outer diameter of from about 900 to about 950 pm.
  • each hollow fibre may be at least 1cm. In some examples, the length of each hollow fibre may be at most 10m . In some examples, each hollow fibre has a length from 1cm to 10m. In some examples, each hollow fibre has a length from 3cm to 5m. In some examples, each hollow fibre has a length from 10cm to 2m.
  • the hollow fibres may be produced by any method, many of which are known in the field. For example, melting spinning, solution spinning, wet spinning, gel spinning, dry-wet spinning, liquid crystal spinning, dispersion spinning, reaction spinning and electrospinning.
  • the hollow fibres may be fibres as described in Luetchford, Kim A., et al. “Next generation in vitro liver model design: Combining a permeable polystyrene membrane with a transdifferentiated cell line.” Journal of membrane science 565 (2016): 425-438, which is incorporated herein in its entirety.
  • the perfusion module may include at least 1 hollow fibre.
  • the perfusion module may include from 1 to 50000 hollow fibres.
  • the methods described herein may include initial sterilisation and/or wash steps.
  • the perfusion module, tubing and other components of the perfusion system may be sterilised.
  • sterilisation may be by autoclaving and/or by application of a sterilisation composition, for example, application of 70% ethanol.
  • the perfusion module without hollow fibres included and reservoir may be autoclaved prior to being connected to the perfusion system.
  • ethanol may be pumped through the system in order to sterilise the component parts of the system.
  • the sterilisation composition may be pumped (or perfused) through the system for at least 30 minutes. For example, at least 30, 40, 50, 60 minutes or more.
  • the methods may include a wash step.
  • the wash step may include removing any sterilisation composition from the system. For example, by draining the sterilisation solution from the system.
  • the wash step may then further include pumping a culture media through the system so as to saturate the system and contact all component parts with the culture media.
  • the cell culture may then be maintained in the system.
  • the culture media used for the wash step may be replaced with a further culture media as described herein. For example, a proliferation media as described herein.
  • the methods described herein include a step of seeding cells onto the hollow fibres in the perfusion module.
  • the cells Prior to seeding, the cells may be pre-cultured.
  • the cells may be pre-cultured outside of the perfusion system to form a cell suspension (seeding culture.
  • the cell suspension may be formed by first washing cells in a buffer, such as phosphate buffered saline (PBS).
  • the buffer may include additional components such as a cell detachment agent .
  • a cell detachment agent may be, for example, an enzyme such as trypsin, trypLE or nattokinase.
  • the cell detachment agent may be a composition, for example, acctuase.
  • the buffer may include at least 0.05% trypsin.
  • the buffer includes about 0.25% trypsin. Additional components may then be added to the buffer including the cells.
  • EDTA may be added to the buffer and cells.
  • the buffer and cells may then be incubated for a period of time. For example, at least 3 minutes. For example, about 5 minutes.
  • a portion of the cells in the buffer may be mixed with a volume of culture media that includes a growth promotion agent, such as serum, for example, foetal bovine serum (FBS).
  • a growth promotion agent such as serum, for example, foetal bovine serum (FBS).
  • FBS foetal bovine serum
  • the serum may help to neutralise the cell detachment agent.
  • growth promotion agents include growth factors (e.g. recombinant growth factors), bovine ocular fluid, sericin protein, earthworm heat inactivated coelomic fluid.
  • the culture media may be a serum free culture media and may optionally include additional components depending on the cells being cultured.
  • serum free culture medias include those commercially available from ThermoFisher, Lonza Bioscience and Merck. The number of cells in the volume of the culture media may then be determined. For example, using a cell counter or other suitable methods.
  • the volume of culture media may be centrifuged to pellet the cells therein.
  • the cell pellet may then be resuspended in a culture media, for example, a proliferation media as described herein, to form the cell suspension.
  • the cell suspension may then be applied to the perfusion module and the hollow fibres therein.
  • the volume applied to the perfusion module may be a volume that at least partially fills the extra-capillary space (ECS).
  • the remaining volume of the perfusion module may be filled with further culture media (not including cells).
  • the volume of cell suspension may be less than the ECS.
  • the cell suspension may be continuously circulated through the perfusion module and therefore continually contacted with the hollow fibres. Continuously circulating the cell suspension may allow for increased seeding efficiency.
  • the number of cells to be applied to the perfusion module may be determined by the outer surface area of the hollow fibres. For example, at least about 1000cells/cm 2 may be applied to the perfusion module. In some examples, around 25,000 cells/cm 2 may be applied to the perfusion module. For example, cells may be added to each square centimetre of each hollow fibre at a concentration of at least 100 cells/cm 2 . Cells may be added to each square centimetre of the outer surface area of all of the hollow fibres at a concentration from 100 cells/cm 2 up to 500,000 cells/cm 2 or more.
  • the number of cells may be determined based on the number of cells used or seeding a 2-dimensional cell culture system.
  • the number of cells applied may be at least 0.1 -fold greater than that used to seed cells onto a flat sheet (2D) cell culture system made of the same material as the hollow fibres.
  • the perfusion module may be maintained at an angle from 0 to 90° from horizontal for a time period sufficient to allow the cells to attach or adhere to the outer surface of the hollow fibres. Maintaining the perfusion module at an angle as described herein may allow for the initial attachment of the cells and thus avoid uneven distribution of cells on the outer surfaces of the hollow fibres.
  • the angle is about 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14,
  • the module is maintained at an angle of between 0 and 30 degrees from horizontal. For example, between 5 and 20 degrees from horizontal. For example, between 8 and 10 degrees from horizontal. For example, between 8.5 and 9.5 degrees from horizontal. In some examples, the angle is about 9.2 degrees. That is to say that the hollow fibres in the module are maintained at the selected angle.
  • the hollow fibres may be any shape and the angle may be defined in relation to an axis through the hollow fibres. For example, for a tubular substantially straight hollow fibre, the angle from horizontal may be taken as the angle between the horizontal plane and the axis running longitudinally through the hollow fibre (i.e. the axis is the same as the lumen of the hollow fibre).
  • the hollow fibre may be spiral, arcuate or non-linear.
  • the angle may be defined by an offset from the plane generally parallel to the horizontal plane. That is to say that when the perfusion module is placed in the horizontal plane, the perfusion module is then offset to the horizontal plane and ergo offsetting the fibres therein from the horizontal plane.
  • the hollow fibres may not be uniform in shape and as such, reference to an angle from horizontal refers to an angle taken from a specific point of the perfusion module or hollow fibres (i.e. the centre point of the longitudinal axis running through the perfusion module or hollow fibres) and as such the angle may not be the same for all parts of the hollow fibres.
  • the perfusion module may be maintained at an angle as described herein for a time period of at least 10 minutes. In some examples, the perfusion module may be maintained at the angle for the entirety of the incubation period and/or culturing (proliferation and/or differentiation).
  • the perfusion module including the cells seeded therein, may then be cultured (incubated) for a time period to allow for further attachment and initial growth of the cells.
  • the perfusion module and cells may be cultured (incubated) for a time of at least 2 hours. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours. In some examples, the perfusion module and cells may be cultured (incubated) for a time of about 3 hours.
  • the perfusion module and cells may be cultured (incubated) under conditions (i.e. temperature and atmosphere) suitable to sustain viable cells and/or allow growth of the cells. For example, for mammalian cells, the perfusion module and cells may be cultured (incubated) at 37°C in an atmosphere including 5% CO2.
  • the perfusion module may be rotated during the initial attachment stage (seeding) and/or incubation time. In some examples, the perfusion module and cells are rotated intermittently. In some examples, the perfusion module and cells are rotated at a speed of between 0 to 30 rpm. In some examples, the perfusion module and cells are rotated at a speed of about 12 rpm. In some examples, the perfusion module and cells are rotated for 1 second to about 30 minutes every 10 seconds to 30 minutes. In some examples, the perfusion module and cells are rotated for about 30 seconds every 5 minutes. That is to say that the perfusion module is rotated for a period of 30 seconds and then remains stationary for 5 minutes before being rotated again. The perfusion module may be rotated for at least 1 hour.
  • the perfusion module may be rotated continuously.
  • the perfusion module may be continuously rotated at a speed of between 0 and 30 rpm.
  • the perfusion module is continuously rotated at a speed between 0 and 4 rpm.
  • the perfusion module is continuously rotated for at least 3 hours. For example, about 3.5 to about 5 hours.
  • Rotation of the perfusion module and cells may be performed by any suitable means.
  • a tube rotator such as a MACSmixTM available from Miltenyi Biotech.
  • Rotation of the perfusion module and cells may be used to exert a centrifugal force on the cells.
  • the centrifugal force may be more than 0.001 N.
  • the centrifugal force may be at least 0.001 N.
  • the centrifugal force may be between 0 and 50 N.
  • the application of centrifugal force may help to move the cells through the perfusion module and bring the cells into contact with the hollow fibres. Without being bound by theory, this may help to improve the attachment efficiency of the cells to the hollow fibres.
  • Intermittent rotation of the module may help introduce motive centrifugal forces to free-floating cells which may improve mixing. The increased mixing may help provide an even distribution of cells across hollow fibre surfaces upon contact and attachment.
  • the force applied by rotation may be affected by the positioning of the perfusion module in relation to the axis of rotation.
  • the perfusion module may be positioned so as to have an offset between the mid-point of the axial centreline of the perfusion module and the axis of rotation.
  • the centreline of the perfusion module refers to the plane running axially through the centre of perfusion module longitudinally as shown by the cross in Figure 6.
  • the mid-point of the centre line refers to the distance halfway along the centreline.
  • Providing an offset in combination with the rotation described above may help to mix the cells within the perfusion module. Without being bound by theory, the mixing imparted by the offset and rotation may increase the surface area of the hollow fibres that is contacted by the cells and may therefore improve the attachment efficiency of the cells to the hollow fibres
  • Maintaining the perfusion module and/or rotation may help improve cell attachment and/or increase the number of viable of cells (i.e. viable cells attached to the hollow fibres). For example, increase the attachment efficiency in comparison to methods not including a maintaining step and/or rotation as described herein 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.
  • Cell viability may be measured using any suitable means known in the field. For example, cell viability may be measured by direct measurement methods. Direct, measurement methods include attaching the cells to the hollow fibres.
  • the fibres including attached cells Prior to culturing (i.e. proliferation or differentiation) the fibres including attached cells are removed and the cells attached to the fibres are removed from the fibres (i.e. detached) by known means. For example using a detachment agent. The number of viable cells that have been detached from the fibres may then be determined. The number of viable cells may be determined by cell counting methods such as flow cytometry or FACS. For example, using a cell counter such as Chemometec NC-200. The total number of attached cells and/or the total number of viable cells may be used to determine an attachment efficiency.
  • cell viability after attachment may be measured by indirect measurements methods.
  • the perfusion module may be drained of growth media and the total cells in the drained growth media are quantified (including non-viable and viable cells) using cell counting methods as described above.
  • the perfusion module After rotating the perfusion module and cells, the perfusion module is connected to the perfusion system, for example, by connecting the tubing to each end of the perfusion module and any sideports that may be included.
  • Cell culture media as described herein may then be perfused (pumped) through the perfusion system and the lumens of the hollow fibres.
  • the cell culture media may be a proliferation media as described herein.
  • the culture media may be pumped from either end of the perfusion module.
  • the perfusion module is maintained at an angle from O to 90°. For example, 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,
  • culture media may be pumped from the lower or upper end to the opposing end.
  • culturing includes maintaining the perfusion module and the hollow fibres with cells attached thereto at an angle of 0° from horizontal.
  • the cells may be cultured under suitable conditions for proliferation of the cells. For example, using the conditions as described herein, which may be selected depending on the type of cells being cultured. For example, for mammalian cells may be cultured (incubated) at 37°C in an atmosphere including 5% CO 2 .
  • the culture media may be changed to a second culture media.
  • the culture media may be changed from a proliferation media to a differentiation media as described herein.
  • cells may be striated.
  • the cells may form myotubes.
  • the cells may form striated myotubules along each channel.
  • the mechanical force from sheer stresses caused by fluid flowing in the ECS and/or the lumens may help the striation of the cells.
  • the second culture media may also be changed to a further media.
  • the further media may be the same as the first culture media (e.g. switching from a differentiation media to a proliferation media) or may be a different culture media.
  • the culture media may be switched from a first, second or further media any number of times.
  • the culturing conditions may be changed depending on the media being perfused through the system.
  • the cells may be cultured in any of the first, second and/or third culture media for at least 3 hours.
  • cell may be cultured for at least 1 day.
  • the cells are continuously cultured. This may provide a system that provides a high number of cells.
  • the cells may be harvested from the perfusion module.
  • Cells may be harvested by removing the cells from the hollow fibres.
  • the cells may be harvested attached to the hollow fibres, for example, by extracting the hollow fibres from the perfusion module with the cells attached thereto.
  • the cells may be harvested when the cells reach a confluence of at least 60%.
  • cells may be harvested after proliferation and/or differentiation, for example, after culturing in proliferation media for a time period as described herein. If culturing includes the use of differentiation culture media, then cells may be harvested when the cells reach a predetermined protein density.
  • the harvested cells or the harvested cells and hollow fibres may be used to produce a comestible product.
  • the cells and hollow fibres may be formed into a meat analogue.
  • a meat analogue (which may also be referred to as cultured or in vitro meat) refers to a food product that is not produced by the slaughter of an animal but has structure, texture, aesthetic qualities, and/or other properties comparable or similar to those of slaughtered animal meat, including livestock (e.g., beef, pork), game (e.g., venison), poultry (e.g., chicken, turkey, duck), and/or fish or seafood substitutes/analogues.
  • livestock e.g., beef, pork
  • game e.g., venison
  • poultry e.g., chicken, turkey, duck
  • fish or seafood substitutes/analogues e.g., fish or seafood substitutes/analogues.
  • the term refers to uncooked, cooking, and cooked meat-like food products.
  • the cells or cells and hollow fibres may be configured to mimic the taste, texture, size, shape, and/or topography of a traditional slaughtered meat.
  • multiple sets of harvested cells or cells and hollow fibres cells may be combined in order to form a structure similar to a cut of meat or a portion-sized product.
  • bound together or compressed together For example, harvested cells or cells and hollow fibres may be bound together by an edible adhesive such as transglutaminase.
  • the harvested cells or cells and hollow fibres may have further agents added in order to make the sensory properties, such as texture, taste, smell and visual properties, more similar to a meat.
  • agents added such as texture, taste, smell and visual properties, more similar to a meat.
  • one or more of fats, texturisers, bulking agents, thickeners, preservatives, flavour enhancers, antimicrobial agents, pH modulators, desiccants, vitamins, minerals, metals, salts, sweeteners, curing or pickling agents, colouring agents, or any combination thereof may be added to the cells or cells and hollow fibres. Additional agents may be dispersed through the cells or cells and hollow fibres via the lumens of the hollow fibres.
  • a meat analogue including cells or cells and hollow fibres produced the methods as described herein.
  • Any meat analogue produced may have the dimensions of a whole cut of meat. For example, it may have at least one dimension (i.e. at least one of length, width or thickness) that is at least 10cm. The thickness or diameter of such a meat analogue may be up to 50cm.
  • the cells may be removed from the hollow fibres. That is to say that the cells may be recovered from the hollow fibres.
  • the cells may be recovered by applying a cell-support specific agent to the hollow fibres.
  • a cell detachment agent may be any agent that is capable of detaching the cells from the hollow fibres.
  • the cell detachment agent may be an enzyme.
  • trypsin, trypLE or nattokinase In specific examples, the cell detachment agent is nattokinase.
  • Nattokinase is a fibrin-specific enzyme derived from fermented soybeans.
  • the nattokinase may be foodgrade nattokinase.
  • the cell detachment agent may be added to the hollow fibres that have been manipulated to be a flat sheet , for example, unrolled or may be applied to intact hollow fibres.
  • the cell detachment agent may be applied at a concentration of at least 10 mg/ml.
  • the cell detachment agent may be applied at a concentration of at least 10, 20, 30, 40, 50, 60 or 70 mg/ml.
  • the cells are removed from the hollow fibres and suspended in a composition including the cell detachment agent. The recovered cells may then be separated from the composition, for example, by centrifugation or other known methods.
  • the recovered cells may then be used to produce a comestible product.
  • the cells may be processed into a product such as a meat analogue.
  • the cells may be subjected to similar processes as used for producing products such as sausages or processed meats products, for example, reconstituted meats such as baloney.
  • the cells may be emulsified, ground, or minced and then formed into a product that resembles a cut of meat or a meat product.
  • processed cells may be moulded or shaped using any known methods.
  • the cells may have agents added to help processing, such as fats, binders, or texturisers added.
  • the cells may also have additional agents added in order to make the sensory properties, such as texture, taste, smell and visual properties, more similar to a meat.
  • additional agents added such as texture, taste, smell and visual properties, more similar to a meat.
  • one or more of fats, texturisers, bulking agents, thickeners, preservatives, flavour enhancers, antimicrobial agents, pH modulators, desiccants, vitamins, minerals, sweeteners, salts, metals, curing or pickling agents, colouring agents, or any combination thereof may be added to the cells.
  • nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
  • Fibers were manufactured in-house using a non- biodegradable proprietary polymer, NMP as the solvent and H2O as the non-solvent.
  • the fibers used in the system described here are 1.05mm outer diameter with a 600-700 pm lumen diameter.
  • the fibers are porous with pore diameters measuring 2.28 pm ⁇ 1.5 pm (mean ⁇ standard deviation). This is designed to separate cells from the media feed in the fiber lumen, replicating vasculature of tissues. Fibers can also be purchased from membrane suppliers such as Pall.
  • the modules used in this study are made from 1 mm thick borosilicate glass with 2 side ports ( Figure 2A).
  • the fibers in the module described here has 3x fibers with a combined outer surface area of 6.54 cm 2 which is the equivalent of roughly half of a well of a 6-well plate.
  • a pre-treatment step is carried out using proliferation medium (DMEM + 10% FBS + 1% P/S).
  • the wash medium is replaced with a reservoir containing 10 ml of proliferation medium (DMEM + 10% FBS + 1% P/S) and pumped at 30 ml/h overnight.
  • the wash medium is replaced with a reservoir containing 10 ml of proliferation medium (DMEM + 10% FBS + 1% P/S) and pumped at 30 ml/h overnight.
  • C2C12 cells a separate pre-treatment step may be carried out.
  • the seeding protocol presented below also serves to pre-culture the module with cell culture medium prior to cell growth. Should a more extensive pre-culture be required then this should be carried out prior to seeding the module by draining the wash media from the system, replacing with growth media and permeating this through the module for few hours. See section 7.5.2.1 for details on replacing media in the system.
  • the number of cells to use in the seeding step should be empirically determined for your desired cell type.
  • the bioreactors described here are seeded at a For C2C12 cells, the bioreactors are seeded at a 4-fold higher cell density that used in 2D tissue culture plastic for a 7 day culture.
  • the reactor module needs to be orientated and maintained at a set angle following the addition of the cell suspension, to prevent an uneven distribution of cells due to gravity settling.
  • the module should remain at the selected angle for at least 10 minutes (specific to initial attachment time of C2C12s) prior to attaching to a tube rotator.
  • a tube rotator (Miltenyi Biotech MACSmixl) is used to rotate the modules intermittently at 12 rpm for 30 seconds with a 5 minute pause between rotation periods.
  • the modules can be drain using a 27G needle to introduce air or instead the side port end caps are removed and the module drained by gravity.
  • the fibers used in the research system described here are set to permeate at ⁇ 80 pl/hr, with an 800 pl/hr feed rate.
  • a metabolic analyser (Roche CEDEX) may be used to measure the concentrations of glucose, lactate, glutamine and ammonia. Kits are available from various suppliers that can quantitate these factors from media (see Table 1 for those used in this study).
  • Injection ports can be added to the permeate and retentate tubing and media sampled via a 27 G needle and syringe, and media can be sampled from the media reservoir bottle. This gives nutrient and metabolism information for the input and both output streams. Sampling should be carried out in a laminar flow hood. Sterilize injection ports before sampling by holding an ethanol soaked blue roll against the port for >30 sec.
  • Imaging 7.4.1 After excision dip the fibers into PBS to wash and use scissors to cut them into smaller lengths into a 24-well plate. Add 400 pl 4% paraformaldehyde (in PBS) and incubate at RT for 20 min.
  • C2C12 immortalised murine myoblast cell line C2C12 as a model skeletal muscle cell line and primary, adult rat skeletal muscle cells, RSkMCs (Sigma-Aldrich, R150-05a).
  • the C2C12s were cultured with DMEM (Sigma Aldrich D5796) supplemented with 10% v/v foetal 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).
  • the RSkMCs were cultured with Rat Skeletal Muscle Cell Growth Medium (Sigma-Aldrich, R151-500). During culture and bioreactor runs, cells were incubated at 5% CO2 and 37 °C. Cell viability was assessed using trypan blue exclusion with 0.4% or 0.04% trypan blue solution (Sigma-Aldrich, T8154) and haemocytometer cell counts.
  • the hollow fibre bioreactor system consisted of a Watson-Marlow 205U peristaltic pump with orange-white colour coded pump tubing, silicone tubing (i.d. 0.8 mm, L/S 13 Platinum-Cured, Masterflex 96410) and a feed bottle with a Whatman Hepa-vent filtration unit (Sigma-Aldrich, WHA67235000).
  • Optimisation of the system included the use of in-line dissolved oxygen sensors (PreSens flow through cell for oxygen, FTC-SU-PST3-S) in conjunction with PreSens Fibox 4 oxygen reader.
  • the hollow fibres used in the system were initially 10% PLGA followed by porous, hydrophilic polystyrene hollow fibres produced inhouse via dry-wet spinning, developed by Luetchford et al. 2018.
  • the move to polystyrene hollow fibres, referred to as PX40 was made after biocompatibility of RSkMCs was tested on flat sheet membranes and compared to the control of tissue culture plastic, TCP using a resazurin-based assay to determine metabolic activity, as a proxy for cell growth.
  • Reactors were used with either 2 or 3 hollow fibres and a total feed flow rate of 800 pl h-1. Reactor setup and operation was performed via the method in Storm et al. 2016, with a 3 h attachment period and dynamic seeding using a MACSMix rotator.[2]
  • the inoculation cell number represents the number of cells placed in the reactor at day 0
  • the seeded cell number (NO) is the starting cell number and is based on an assumed seeding efficiency of 10% and NF is the final, total number of cells present at the end of the run (viable and non-viable).
  • PLGA poly(lactic-co-glycolic acid
  • PX40 polystyrene based polymer with 40% w/w microcrystalline sodium chloride.
  • HFB module ran dry (cells not exposed to nutrient supply), suspected back pressure issues; b Pump stopped at day 3 due to power outage (4 days of no media circulation followed); and c Trypan blue exclusion assay during cell counts indicated cells were non-viable.
  • the number of cells obtained from the bioreactor systems can theoretically be increased by increasing the duration of the proliferation phase and time per reactor run and the number of hollow fibres within the reactor module.
  • the number of hollow fibres dictates the surface area available for cell attachment and growth and a confluent surface dictates the need for transferring to a larger reactor system, known as passaging.
  • the NF values are suspected to be an under-estimate of the total, final number of cells due to the presence of large cell aggregates during haemocytometer counting.
  • the presence of aggregates is suspected to result from the method of cell dissociation from the fibre scaffolds, trypsin is currently used.
  • the desired cell number (suspension concentration of cells per mL and the corresponding volume in mL to reach target seeding density).
  • a suspension volume less than or equal to the ECS volume was first injected into the reactor and then topped up with fresh media to avoid losses of cells if not all of the ECS volume was not injected into side port of reactor from the original seeding suspension volume.
  • the module was mounted in a specially designed saddle clip on rotor at specific angle (in this case, 9.2 degrees).
  • Constant rotation was applied to ensure good distribution of cells across the fibre bundle.
  • the module was removed; unattached cells removed by draining the growth medium from the side port and replaced with fresh growth medium and connected to a continuous, dynamic feeding system following seeding.
  • the seeding regime comprised of rotating the perfusion module inclined at 9.2 degrees, rotating continuously at 0.3 RPM, the module was offset by 150mm from the axis of rotation.
  • Viability of cells was determined by either a direct or indirect method of cell viability measurement.
  • a - Proliferation experiments are those which are run for 5-7 days. The cells are not removed from the fibres directly after seeding, hence only an indirect measurement was used.

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Abstract

La présente invention concerne un procédé de culture de cellules musculaires pour un produit comestible, le procédé consistant à ensemencer des cellules musculaires sur une ou plusieurs fibres creuses poreuses, à maintenir le module de perfusion dans une orientation permettant à une ou plusieurs fibres creuses poreuses de former un angle compris entre 0 et 90 par rapport à l'horizontale pendant une période suffisante pour permettre l'attachement initial des cellules musculaires à une surface externe de chacune des fibres creuses poreuses, à faire tourner le module de perfusion, à connecter le module de perfusion à un système de bioréacteurs à perfusion, et à cultiver les cellules musculaires attachées à la surface externe des fibres creuses poreuses. L'invention concerne également des produits comestibles produits selon de tels procédés.
PCT/GB2023/052165 2022-08-18 2023-08-17 Procédé de culture de cellules musculaires avec bioréacteur à perfusion WO2024038281A1 (fr)

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