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WO2024134176A1 - Système de biotraitement - Google Patents

Système de biotraitement Download PDF

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Publication number
WO2024134176A1
WO2024134176A1 PCT/GB2023/053301 GB2023053301W WO2024134176A1 WO 2024134176 A1 WO2024134176 A1 WO 2024134176A1 GB 2023053301 W GB2023053301 W GB 2023053301W WO 2024134176 A1 WO2024134176 A1 WO 2024134176A1
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WIPO (PCT)
Prior art keywords
cell
cells
density
cell culture
adherent cells
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PCT/GB2023/053301
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English (en)
Inventor
Ajay THAKARAN
Che Connon
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Cellularevolution Ltd
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Cellularevolution Ltd
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Publication of WO2024134176A1 publication Critical patent/WO2024134176A1/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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Definitions

  • This invention relates generally to a bioprocessing system. More specifically, although not exclusively, this invention relates to a system for continuous bioprocessing of adherent cells, and uses thereof. The invention also relates to a method for continuous bioprocessing of adherent cells.
  • Methods of producing cultured, or cultivated, meat by cultivating animal cells are also being developed as an alternative to farming animals for food.
  • the first phase of growth is referred to as the lag phase, where the cells are seeded into a growth chamber (e.g. onto a support or into the culture media to grow as cell aggregates), they take time to adapt to their new environment, and prepare for rapid growth.
  • the log phase which follows is a period in which the cells grow exponentially and consume the nutrients in the cell culture media.
  • the growth media is spent, or when the cells reach confluency, the cells enter the stationary phase. In this phase, cell proliferation is greatly reduced or may stop entirely.
  • traditional cell culture methods the cells are harvested in bulk after the cells enter the stationary phase. The spent cell culture media is removed from the cell culture. An enzyme, such as trypsin, is then added to detach the cells, and the cells are harvested from the culture vessel. The process of seeding cells, and growth in lag phase is then repeated before log phase growth can be achieved.
  • Such traditional cell culture methods are batch processes.
  • a further disadvantage of current methods for culturing adherent cells is the need to increase the reactor size, and the associated cost, in order to yield a sufficient number of cells.
  • the present invention relates to a system for continuous bioprocessing of adherent cells.
  • the system includes a cell growth chamber, a fluid reservoir in fluid communication with the cell growth chamber and a control loop for measuring and regulating the density of adherent cells in the cell growth chamber.
  • the control loop includes at least one sensor that is configured to measure the density of adherent cells in the cell growth chamber and a controller that is configured to automatically regulate the supply of a cell detachment agent from the fluid reservoir to the cell growth chamber in response to the measured density of adherent cells.
  • the system may comprise a cell growth chamber.
  • the system may also comprise a fluid reservoir in fluid communication with the cell growth chamber.
  • the system may also comprise a control loop for measuring and regulating the density of adherent cells in the cell growth chamber.
  • the control loop may comprise at least one sensor configured to measure the density of adherent cells in the cell growth chamber.
  • the control loop may comprise a controller configured to automatically regulate the supply of a cell detachment agent from the fluid reservoir to the cell growth chamber in response to the measured density of adherent cells.
  • a system for continuous bioprocessing of adherent cells comprising: a cell growth chamber; a fluid reservoir in fluid communication with the cell growth chamber; and a control loop for measuring and regulating the density of adherent cells in the cell growth chamber, the control loop comprising: at least one sensor configured to measure the density of adherent cells in the cell growth chamber; and a controller configured to automatically regulate the supply of a cell detachment agent from the fluid reservoir to the cell growth chamber in response to the measured density of adherent cells.
  • the cell detachment agent may be a protease, for example an exogenous protease.
  • the exogenous protease may be an exogenous metal loprotease and/or cysteine protease.
  • the metalloprotease may, for example, be selected from the group consisting of: collagenase and dispase.
  • the metal loprotease may, for example, be collagenase.
  • the collagenase may be selected from the group consisting of: collagenase type I, collagenase type II, collagenase type III, collagenase type IV, collagenase type V, collagenase type VI, and collagenase type VII.
  • collagenase type I to type VII are enzyme compositions comprising collagenase at increasing levels of purity, with collagenase type VII being pure collagenase.
  • the metalloprotease may be a dispase.
  • the dispase may be dispase I or dispase II.
  • the cysteine protease may be of the CA clan according to the MEROPS classification scheme (https://www.ebi.ac.uk/merops/).
  • the cysteine protease of the CA clan may be selected from the group consisting of ficin, papain, bromelain, cathepsin K and calpain. More suitably, the cysteine protease of the CA clan may be ficin and/or papain.
  • the at least one sensor may be configured to directly measure the density of adhered cells.
  • the at least one sensor may, for example, measure the density by a means selected from the group comprising: microscopy, acoustic resonance densitometry, laser induced fluorescence, fluorescence microscopy, capacitance impedance, turbidity, biomass permittivity probe, Raman probe and cell counters e.g. CCD imaging using Trypan blue.
  • the at least one sensor may be configured to indirectly measure the density of adhered cells.
  • the at least one sensor may, for example, be configured to measure the consumption of one or more of oxygen or glucose.
  • the at least one sensor may be configured to measure the production of adenosine triphosphate.
  • the controller may be configured to receive a measured cell density from the at least one sensor, to compare the measured cell density with a predetermined target cell density and to regulate the supply of the cell detachment agent in response to the comparison.
  • the controller may be configured to increase the supply of the cell detachment agent.
  • the controller may be configured to decrease the supply of the cell detachment agent.
  • the fluid reservoir may comprise the cell detachment agent alone.
  • the fluid reservoir may comprise a cell culture medium.
  • the fluid reservoir may, for example, comprise a mixture of a cell culture medium and the cell detachment agent.
  • the system may comprise a collection container.
  • the collection container may be fluidly coupled to the cell growth chamber for collecting non-adherent or detached cells suspended in cell culture medium.
  • the control loop for measuring and regulating the density of adherent cells in the cell growth chamber may be a first control loop.
  • the system may comprise a second control loop for assessing the quality of the non-adherent or detached cells.
  • the second control loop may comprise at least one sensor for monitoring a characteristic of the non-adherent or detached cells.
  • the at least one sensor of the second control loop may be, for example, configured to determine a phenotype of the non-adherent or detached cells.
  • the second control loop may comprise at least one sensor for monitoring a characteristic of a cell culture medium in which the non-adherent or detached cells are suspended.
  • the at least one sensor of the second control loop may be, for example, configured to quantify a nutrient or other component of the cell culture medium.
  • the system may be a continuous bioprocessing system.
  • the continuous bioprocessing system allows the culturing and harvesting of adherent cells with minimal human intervention and increased productivity per unit volume.
  • an enzyme such as trypsin
  • a harvesting step in order to detach the cells, and allow the cells to be harvested from the culture vessel.
  • the continuous bioprocessing system of the present invention includes at least one sensor that is configured to measure the density of adherent cells in the cell growth chamber; and a controller that is configured to automatically regulate the supply of a cell detachment agent from the fluid reservoir to the cell growth chamber in response to the measured density of adherent cells.
  • the cell detachment agent may be supplied to the cell growth chamber throughout the cell culture process (not just at the end of the process) in order to optimise the productivity per unit volume of the cell culture system. The detachment of cells therefore occurs gradually over time and is not one discrete (bulk detachment) event.
  • a method for continuous bioprocessing of adherent cells comprising: measuring the density of adherent cells in a cell growth chamber; and regulating, for example automatically regulating, the density of adherent cells in the cell growth chamber.
  • the step of regulating the density of adherent cells comprises using a controller to automatically regulate the supply of a cell detachment agent from a fluid reservoir to the cell growth chamber in response to the measured density of adherent cells.
  • the step of regulating the density of adherent cells may comprise using at least one sensor to measure the density of adherent cells in the cell growth chamber; and using the controller to receive the measured cell density from the at least one sensor, to compare the measured cell density with a predetermined target cell density and to regulate the supply of the cell detachment agent in response to the comparison.
  • the system for continuous bioprocessing of adherent cells of the present invention is associated with a number of advantages.
  • the system utilises a cell growth chamber that aids proliferation and harvesting of adherent cells with minimal human intervention.
  • the system for continuous bioprocessing of cells is closed and sterile, and/or does not contain serum or other xenobiotics, thereby allowing the cells to be produced using good manufacturing practices.
  • the system further enables the rate of cell detachment to be matched to the rate of cell growth, thereby optimising the recovery of cells during the log phase of growth.
  • the method may be a continuous bioprocessing method.
  • the method may comprise any one or more features or steps relevant to one or more features of the system.
  • a further aspect of the invention provides a computer program element comprising computer readable program code means for causing a processor to execute a procedure to implement one or more steps of the aforementioned method.
  • a yet further aspect of the invention provides the computer program element embodied on a computer readable medium.
  • a yet further aspect of the invention provides a computer readable medium having a program stored thereon, where the program is arranged to make a computer execute a procedure to implement one or more steps of the aforementioned method.
  • a yet further aspect of the invention provides a control means or control system or controller comprising the aforementioned computer program element or computer readable medium.
  • any controller(s), control units and/or control modules described herein may each comprise a control unit or computational device having one or more electronic processors.
  • the controller may comprise a single control unit or electronic controller or alternatively different functions of the control of the system or apparatus may be embodied in, or hosted in, different control units or controllers or control modules.
  • control unit and “controller” will be understood to include both a single control unit or controller and a plurality of control units or controllers collectively operating to provide the required control functionality.
  • a set of instructions could be provided which, when executed, cause said controller(s) or control unit(s) or control module(s) to implement the control techniques described herein (including the method(s) described herein).
  • the set of instructions may be embedded in one or more electronic processors, or alternatively, may be provided as software to be executed by one or more electronic processor(s).
  • a first controller may be implemented in software run on one or more electronic processors, and one or more other controllers may also be implemented in software run on or more electronic processors, optionally the same one or more processors as the first controller. It will be appreciated, however, that other arrangements are also useful, and therefore, the present invention is not intended to be limited to any particular arrangement.
  • the set of instructions described herein may be embedded in a computer-readable storage medium (e.g., a non-transitory storage medium) that may comprise any mechanism for storing information in a form readable by a machine or electronic processors/computational device, including, without limitation: a magnetic storage medium (e.g., floppy diskette); optical storage medium (e.g., CD-ROM); magneto optical storage medium; read only memory (ROM); random access memory (RAM); erasable programmable memory (e.g., EPROM ad EEPROM); flash memory; or electrical or other types of medium for storing such information/instructions.
  • a computer-readable storage medium e.g., a non-transitory storage medium
  • a magnetic storage medium e.g., floppy diskette
  • optical storage medium e.g., CD-ROM
  • magneto optical storage medium e.g., magneto optical storage medium
  • ROM read only memory
  • RAM random access memory
  • Figure 1 is a schematic representation of a system for continuous bioprocessing of adherent cells
  • Figure 2 is a schematic representation of another system for continuous bioprocessing of adherent cells
  • Figure 3 is a schematic representation of a further system for continuous bioprocessing of adherent cells
  • Figure 4 is a schematic representation of yet another system for continuous bioprocessing of adherent cells
  • Figure 5 is a schematic representation of a cell growth chamber
  • Figure 6 is a flow chart illustrating the use of a system for continuous bioprocessing of adherent cells.
  • Figure 7 is a schematic representation of a yet further system for continuous bioprocessing of adherent cells.
  • a “support” as referred to herein is any surface, which is suitable for supporting biological material, for example a cell, cell population, cell culture, tissue, or a fluid or other biological material or composition, for example as described herein.
  • a support may be referred to as a surface, may be suitable for hosting a process or reaction, for example, growth and development of a cell or organism, cell population, cell culture, or tissue.
  • a “gel” is a semi-solid, jelly like substance, which does not flow when in the solid state.
  • a gel comprises a 3D cross linked network which provides the gel its semi-solid structure.
  • a gel may be a hydrogel, which comprises a network of insoluble but hydrophilic polymer chains.
  • a gel may be defined in terms of its viscoelasticity or rheological properties.
  • a “buffer” is a solution which can resist significant changes in pH upon the addition of small amounts of acid or alkali.
  • a buffer is a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid.
  • cell viability refers to the ability of a cell to remain metabolically active for growth and function.
  • a cell “culture period” is a period of time during which cells are maintained in cell culture, under conditions suitable for growth.
  • cell culture medium refers to a nutritive solution for cultivating live cells so as to allow the cell to proliferate.
  • the term "contacting” refers to causing two or more items to come into contact with each other.
  • the items may be two or more of cells, a substrate, a fluid, and a purified mucin or biosimilar mucus, suitably as defined herein.
  • Contacting may include causing or placing two or more of the above items into close physical relationship and/or touching with each other. Contacting may sometimes be referred to herein as “exposing”, for example exposing cells to a cysteine protease shall be therefore understood as contacting cells with a cysteine protease.
  • adherent cells refers to a homogenous or heterogeneous population of cells which are anchorage dependent, i.e. which require attachment to a support or to other adherent cells (e.g. in the form of a cell aggregate) in order to grow in vitro.
  • cell aggregate may also be referred to as a multicellular aggregate herein.
  • An aggregate refers to e.g. a ball, cluster, layer etc of cells. It refers to a plurality of adjoining or interconnected cells.
  • a cell aggregate may be formed from e.g. at least 10 adjoining cells (wherein each cell is in direct contact (in other words touching) with at least one other cell within the aggregate).
  • the aggregate may comprise at least 10, at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , or at least 10 9 etc adjoining cells.
  • the adjoining cells are interconnected.
  • adjoining refers to cells that are connected to each other in a manner that forms an aggregate of cells.
  • the adjoining cells retain the aggregate form when placed in a solution such as cell culture medium.
  • Adjoining cells may be in direct contact e.g. wherein they adhere to or touch each other in a manner that forms an aggregate of cells.
  • adjoining cells may be connected indirectly in a manner that forms an aggregate of cells, such as by virtue of the presence of a matrix, support or scaffold (e.g. an extracellular matrix), wherein the matrix, support or scaffold connects the adjoining cells into the aggregate.
  • a matrix, substrate or scaffold may connect adjoining cells, to form an aggregate.
  • the structure may also facilitate or maintain aggregate formation.
  • the structure may be naturally derived or synthetic.
  • the structure may be a synthetic or natural polymer.
  • the structure is biodegradable.
  • the structure may, for example be a polymer comprising polylactic acid (e.g. poly(lactic acid-co-caprolactone) (PLACL)), collagen or nylon.
  • PLACL poly(lactic acid-co-caprolactone)
  • the cells are adjoined via an extracellular matrix (ECM) in a manner that forms a multicellular aggregate.
  • ECM extracellular matrix
  • a further example of a suitable structure is an Alvetex® polystyrene scaffold for 3D cell culture.
  • Other structures may comprise collagen, gelatin, alginate, cellulose, glass, or matrigel, etc.
  • the structure may also be a nylon mesh.
  • the aggregates may be structure-free.
  • Appropriate methods for cell culture with or without a structure are well known in the art.
  • the adjoining cells are interconnected.
  • interconnected refers to cells that are in direct contact with each other and are physically connected e.g. by intercellular connections (e.g. by one or more cell junction(s) (also known as intercellular bridge(s))).
  • Cell junctions are made up of multiprotein complexes that provide contact between neighboring cells or between a cell and the extracellular matrix. Cell junctions are especially abundant in epithelial tissues. Cell junctions enable communication between neighbouring cells.
  • the cell aggregate may be any group of adjoining cells, for example, it may be in the form of a tissue or an organ (e.g. an animal or plant tissue or organ, or a synthetic/artificial tissue or organ i.e. tissue engineered tissue or organ).
  • tissue or an organ e.g. an animal or plant tissue or organ, or a synthetic/artificial tissue or organ i.e. tissue engineered tissue or organ.
  • suitable animal tissues or organs include skin, cornea, muscle, liver, and heart tissues or organs. Such tissues or organs may be obtained directly from a living animal. Methods for isolating appropriate multicellular aggregates from animals are well known in the art.
  • suitable plant tissues or organs that are obtained from a living plant
  • suitable plant tissues or organs include cells or tissues derived from the endoderm, mesoderm and ectoderm germ layers, mesophyll tissue, xylem tissue and phloem tissue, leaf, stem, root, and reproductive organs. Methods for isolating appropriate multicellular aggregates from plants are well known in the art.
  • suitable synthetic tissue or organs include any cellular tissues or organs that have been generated or propagated in vitro or ex vivo.
  • Non-limiting examples include cellular spheres, spheroids, organoids or micro-tissues.
  • the cell aggregate may be attached to a surface of a receptacle (e.g. culture vessel) in which they were seeded and/or grown in vitro.
  • the cell aggregate comprises a plurality of adjoining (e.g. interconnected) cells, wherein the cells form a tissue, a cell layer, a spheroid, an organoid or any combination thereof.
  • the cells in the cell aggregate are all of the same type. For example, they may all be brain cells, muscle cells or heart cells. In other examples, the cells in the multicellular aggregate are all from the same lineage, e.g. all haematopoietic precursor cells. In some examples, the cells are stem cells, for example, neural stem cells or embryonic stem cells.
  • a multicellular aggregate comprises homogeneous or heterogeneous cell types.
  • cell growth it is meant the division, or proliferation, of the seeded cells.
  • seeding cells it is meant the application of an initial cell population to the cell culture media or support.
  • seeding cells are the cells initially applied to the support.
  • Log phase of growth is the logarithmic or exponential phase of cell growth where the cells are actively proliferating and the cell density is increasing. The log phase typically follows the lag phase, and is prior to the stationary phase where growth slows or stops.
  • yield as used herein in relation to the production of cells means the number of cells produced within a volume of medium in a given time period, for example the number of cells per litre per hour.
  • metalloprotease refers to a protease having one or more metal ions in the binding/active site.
  • metalloprotease examples include collagenase and/or dispase.
  • cyste protease is intended to describe a protease that possesses a highly reactive thiol group of a cysteine residue at the catalytic site of the enzyme. Cysteine proteases are known in the art, and may be referred to herein as “thiol proteases” or “sulfhydryl proteases”. Many superfamilies of cysteine proteases are known in the art.
  • the cysteine proteases may be of the CA clan (also sometimes referred to as Papain-like proteases).
  • Papain-like proteases share a common catalytic dyad active site featuring a cysteine amino acid residue that acts as a nucleophile.
  • the cysteine protease of the CA clan may be selected from the group consisting of ficin, papain, bromelain, cathepsin K and calpain. More suitably, the cysteine protease of the CA clan may be ficin, papain, and/or bromelain. More suitably, the cysteine protease of the CA clan may be ficin and/or papain.
  • papain may be formulated as Performase®.
  • Performase® is powder preparation of purified papain, standardized with Maltodextrin.
  • Papain is an enzyme that is extracted from the green unripe fruit of the papaya tree.
  • Ficin also sometimes referred to as ficain, is a proteolytic enzyme typically extracted from the latex sap from the stems, leaves, and unripe fruit of the American wild fig tree Ficus insipida.
  • the present invention provides a system for continuous adherent cell culture, in which the cells are detached from a support (or from other adherent cells e.g. from a cell aggregate) during the proliferation (or growth) phase, such that confluency may be delayed and cell yield may be increased.
  • the invention is based upon providing a control system for measuring and regulating the density, or confluency, of adherent cells within a cell growth chamber.
  • the system includes at least one sensor that is configured to measure the density, or confluency, of adherent cells in the cell growth chamber and a controller that is configured to automatically regulate the supply of a cell detachment agent, for example a protease, in the cell culture medium in response to the measured density, or confluency, of adherent cells.
  • a cell detachment agent for example a protease
  • the present invention therefore provides a method for continuous growth and detachment of adherent cells.
  • the cells are maintained in both cell culture media and a cell detachment agent which is capable of detaching cells from a support on which the cells grow, or from a cell aggregate, under conditions suitable for cell growth/division.
  • the cells may be detached continuously, meaning that within the system of the invention, the stationary phase of cell growth is not reached as the cell detachment agent present in the cell media continually detaches the cells from the support and/or from other adherent cells e.g. from a cell aggregate.
  • the system and method of the invention are associated with a number of advantages. For example, by matching the cell detachment rate with the cell growth rate, the system and method enable an increase in the yield of cells from the cell culture, by maintaining the cells in the log phase of growth for a longer period of time. In addition, the system and method provides advantages of reducing the amount of resources required to produce a desired yield, and consequently the resource or carbon footprint.
  • the bioprocessing system 10 includes a cell culture chamber 12 (also referred to herein as a cell culture reactor or as a bioreactor), a first fluid reservoir 14 in the form of a pre-reactor, a second fluid reservoir 16 in the form of a supply of a cell detachment agent, e.g. an enzyme such as an exogenous protease, for example wherein the exogenous protease is an exogenous metalloprotease and/or cysteine protease.
  • the exogenous protease may be metal loprotease (for e.g.
  • the bioprocessing system 10 also includes a number of pumps 28a, 28b, 28c.
  • adherent cells refers to a homogenous or heterogeneous population of cells which are anchorage dependent, i.e. which require attachment to a support or to other adherent cells (e.g. in the form of a cell aggregate), in order to grow in vitro.
  • the support may be a cell culture plate, or it may be any other suitable surface, such as a microcarrier (e.g. a support matrix that allows for the growth of adherent cells in bioreactors).
  • Adherent cells attach to the surface or to other cells through an anchorage substrate such as integrin or other cell receptor. It will be appreciated that the surface (e.g.
  • Anchorage dependent cells are typically derived from a multi-cellular organism.
  • the adherent cells may be mammalian cells, for example human, mice rat, rabbit, dog, cat, cow, pig, chicken, goat, horse, etc.
  • Mammalian cells may be derived from any suitable tissue, for example adrenal, bladder, blood vessel, bone, bone marrow, brain, cartilage, cervical, corneal, endometrial, oesophageal, gastrointestinal, immune system (e.g., T lymphocytes, B lymphocytes, leukocytes, macrophages, and dendritic cells), liver, lung, lymphatic, muscle (e.g., cardiac muscle), neural, ovarian, pancreatic (e.g., islet cells), pituitary, prostate, renal, salivary, skin, tendon, testicular, and thyroid.
  • the cells are mammalian cells (e.g., human).
  • the adherent cells may be nonmammalian cells, such as insect or fish or bird cells.
  • the cell may be a prokaryotic cell, for example a fungal cell or a bacterial cell.
  • the adherent cells may be plant cells.
  • the adherent cells may be primary cells or immortalised cells.
  • primary cells may be selected from the group consisting of myocytes, cardiomyocytes, epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, osteoblasts, chondrocytes, adipocytes and mesenchymal stem cells.
  • immortalised cells may be selected from the group consisting of HeLa cells, HEK 293 cells, 3T3 cells, A549 cells, VERO cells, CHO cells, OK cells, C2C12 and Ptk2 cells.
  • the cell may be a disease cell or a disease model cell, for example a cancer cell or a cell in a hyperproliferative state.
  • the cell population comprises cells selected from the group consisting of: skin, muscle, cervical, breast, and prostate cells.
  • the support may be provided in a suitable growth chamber or vessel.
  • a suitable support for use in the present may be selected by the skilled person based upon factors including the type of cells to be cultured, and the desired outcome of the culture. Where necessary, a support may be modified or adapted to support or improve adherence of cells thereto.
  • Sterilization may be performed, for example, by gamma-irradiation, by autoclave, by steam sterilisation (for example in situ steam sterilisation, e.g. by a steam or sterilise in place system), by chemical treatment, for example by washing with alcohol or by ethylene oxide (EtO) gas treatment, or by treatment with aqueous solutions of sodium hydroxide, e.g. up to 5 M aqueous sodium hydroxide at up to 80°C, or by treatment with peroxyacetic acid, e.g. up to 5% peroxyacetic acid at up to 80°C .
  • gamma-irradiation by autoclave
  • steam sterilisation for example in situ steam sterilisation, e.g. by a steam or sterilise in place system
  • chemical treatment for example by washing with alcohol or by ethylene oxide (EtO) gas treatment
  • EtO ethylene oxide
  • peroxyacetic acid e.g. up to 5% peroxy
  • the support may be any surface on which cells may be grown.
  • a support is solid or semi-solid.
  • a support may be 2- dimensional (2D) or 3-dimensional (3D).
  • An example of a 2D surface is a cover slip, or a surface of a culture vessel, such as a tube, a flask, a dish or a plate comprising a plurality of wells.
  • An example of a 3D surface is a scaffold, such as a polystyrene scaffold (e.g. AlvetexTM) or a gel scaffold (e.g. hydrogel).
  • a support may be a slide, chip, plate, flask, vial, film, microstructure (including a groove, well or post), bead, a matrix, a resin, a gel, a film, a membrane, multi-well plate, dual chamber plate, or any other suitable form.
  • a support can be in any suitable shape or configuration, including flat, tubular, curved, spherical, ellipsoid, etc., including composites (e.g. to emulate macro-anatomical structures).
  • a support may comprise a planar surface, upon which a cell population may be provided.
  • a support can be provided or mounted on a porous carrier (e.g. a porous membrane, a mesh, an inorganic grid, a hydrogel, or a combination thereof) to lend structural support thereto.
  • a support may be edible.
  • a support may be rigid or may be elastic.
  • a support may be porous, or may be non- permeable.
  • a material which is not naturally porous can be made porous by methods available to the skilled person, for example sintering, etching, leaching, lithography, or laser micromachining.
  • An example of a porous support may be a gel or a mesh.
  • the support comprises a material selected from the group consisting of: plastic, polymer, glass, and metal.
  • the support is selected from the group consisting of: a bead, a microcarrier, a microfluidic chip, a silicon chip, a microscope slide, a microplate well, a matrix, a resin, a biochip, a multi-well plate, a gel, a film, and a membrane.
  • a material that facilitates cell adhesion may be selected from the group consisting of a polyester, a polypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinyl chloride, a polyvinyl fluoride resin, a polystyrene, a polysulfone, a polyurethane, a polyethylene terephthalate, a cellulose, a glass fiber, a ceramic particle, a matrigel, an extracellular matrix component, a collagen, a poly-L-lactic acid, a dextran, an inert metal fiber, silica, natron glass, borosilicate glass, chitosan, or a vegetable sponge.
  • the cellulose may be cellulose acetate.
  • the extracellular matrix component may be one or more of fibronectin, vitronectin, chondronectin, or laminin.
  • the adherent material is electrostatically charged.
  • the adherent material may be coated with collagen or gelatin.
  • the adherent material may comprise a peptide amphiphile (PA), such as that described in Miotto et al, Developing a Continuous Bioprocessing Approach to Stromal Manufacture, ACS Applied Materials & Interfaces 2017 9 (47), 41131-41142.
  • Adherent cells may attach to the surface through an anchorage substrate such as integrin or other cell receptor.
  • a support may comprise a nonadherent portion.
  • a “growth chamber” refers to any suitable chamber or vessel that is suitable for the growth of adherent cells.
  • a “growth chamber” may therefore have any suitable type of surface and any suitable type of geometry.
  • Appropriate growth chambers are well known in the art and include, but are not limited to, microcarriers, cell culture plates, cell culture containers, reaction vessels, as well as bioreactors. Suitable growth chambers may therefore include, but are not limited to hollow fibre, stirred tank, airlift, bubble column or fluidised bed reaction vessels/bioreactors, or a flexible bag. Accordingly, it is clear that any suitable growth chamber (e.g. a bioreactor) may also be used.
  • the cell growth chamber 12 has an inlet 42 and an outlet 44.
  • the inlet 42 is fluidly connected to the pre-reactor 14 for the supply of a cell culture medium and the outlet 44 is fluidly connected to a collection container or chamber 20.
  • Each of the inlet 42 and the outlet 44 may include a luer-lock connector, or any other hygienic connection, by which they can be fluidly connected to their respective components of the system.
  • cell culture medium refers to a nutritive solution for cultivating live cells so as to allow the cell to proliferate.
  • the cell culture medium may be a complete formulation, i.e. , a cell culture medium that requires no supplementation to culture cells, or may be an incomplete formulation, i.e., a cell culture medium that requires supplementation or may be a medium that may supplement an incomplete formulation or in the case of a complete formulation, may improve culture or culture results.
  • Various cell culture media will be known to those skilled in the art, who will also appreciate that the type of cells to be cultured may dictate the type of culture medium to be used.
  • a cell culture media is selected which does not substantially affect the activity of the selected metalloprotease (such as collagenase and/or dispase), and/or cysteine protease (such as papain and/or ficin).
  • the cell culture medium may be selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Ham's F-12 (F-12), Leibovitz's L-15 medium, RPMI-1640, MesencultTM Basal Medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), Ham's F-10, aMinimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium (IMDM), or any combination thereof.
  • DMEM Dulbecco's Modified Eagle's Medium
  • F-12 Ham's F-12
  • Leibovitz's L-15 medium RPMI-1640
  • MesencultTM Basal Medium MEM
  • Minimal Essential Medium MEM
  • Basal Medium Eagle BME
  • Ham's F-10 Ham's F-10
  • aMEM aMinimal Essential Medium
  • G-MEM Glasgow's Minimal Essential Medium
  • IMDM
  • the media may be selected from the group consisting of 293 SFM, CD-CHO medium, VP SFM, BGJb medium, Brinster's BMOC- 3 medium, cell culture freezing medium, CMRL media, EHAA medium, eRDF medium, Fischer's medium, Gamborg's B-5 medium, GLUTAMAXTM supplemented media, Grace's insect cell media, HEPES buffered media, Richter's modified MEM, IPL-41 insect cell medium, McCoy's 5A media, MCDB 131 medium, Media 199, Modified Eagle's Medium (MEM), Medium NCTC-109, Schneider's Drosophila medium, TC-100 insect medium, Waymouth's MB 752/1 media, William's Media E, protein free hybridoma medium II (PFHM II),
  • the cell culture medium may be serum-free.
  • the cell culture medium may be glucose free.
  • the cell culture medium may comprise serum.
  • Appropriate types and amounts of serum are known e.g. 1% FBS may be used.
  • the supply of cell culture medium 18 is in fluid communication with the pre-reactor 14 such that fresh (i.e. new or unused) cell culture medium can be supplied to the pre-reactor 14 via the pump 28a.
  • the supply of cell detachment agent 16 for example a metalloprotease (e.g. collagenase and/or dispase) and/or a cysteine protease (such as ficin and/or papain), is in fluid communication with the prereactor 14 such that the cell detachment agent can be supplied to the pre-reactor 14 via the pump 28b.
  • the cell detachment agent and the cell culture medium are mixed in the prereactor 14 to provide a target concentration of cell detachment agent in the cell culture medium.
  • the pre-reactor 14 is in fluid communication with the cell culture chamber 12 such that the cell culture medium including the target concentration of cell detachment agent is supplied to the cells within the cell culture chamber 12 via the pump 28c and the inlet 42 (shown in Figure 5).
  • the target concentration of cell detachment agent is selected to ensure that, as the adherent cells grow and proliferate within the cell culture chamber 12, cells are continuously detached from the surface or aggregate and collected, together with spent (or used) cell culture medium within the collection chamber 20.
  • the control loop 22 is provided to ensure that the density or confluency of adherent cells within the cell culture chamber 12 is optimum for further cell growth and proliferation.
  • the sensor 24 is a biomass analysis sensor and is used to assess or determine the density or confluency of the adherent cells.
  • the biomass data in other words the measured or determined density or confluency of the adherent cells, is transmitted or otherwise relayed to the controller 26.
  • the sensor 24 of the control loop 22 is configured to measure the density, or confluency, of the adherent cells within the cell growth chamber 12.
  • the sensor 24 may be configured to directly measure the density of adhered cells within the cell growth chamber 12 and may be selected from the group comprising: microscopy, acoustic resonance densitometry, laser induced fluorescence, fluorescence microscopy, capacitance impedance, turbidity, biomass permittivity probe, Raman probe and CCD imaging (e.g. using Trypan blue) or other cell counters.
  • the sensor 24 may be configured to indirectly measure the density of adhered cells within the cell growth chamber 12.
  • the sensor 24 may, for example measure the consumption of one or more of nutrients by the adhered cells, e.g. the consumption of oxygen or glucose. Additionally, or alternatively, the at least one sensor may measure the production of adenosine triphosphate (ATP) by the adhered cells.
  • ATP adenosine triphosphate
  • the controller 26 is a proportional-integral-derivative (or PID) controller.
  • the controller 26 continuously calculates an error value e(t) as the difference between the desired cell density or confluency and the measured cell density or confluency.
  • the controller 26 acts on the pump 28b to adjust the concentration or activity of the cell detachment agent in the cell culture medium that is supplied to the cell culture chamber 12 from the pre-reactor 14.
  • the controller 26 will act on the pump 28b to increase the supply of cell detachment agent to the pre-reactor 14, thereby increasing the concentration of the cell detachment agent in the cell culture medium that is supplied to the cell culture chamber 12 via the pump 28c.
  • the increased concentration of cell detachment agent will increase the rate at which cells are detached from the surface, thereby reducing the cell density or confluency.
  • the controller 26 will act on the pump 28b to decrease the supply of cell detachment agent to the pre-reactor 14, thereby decreasing the concentration of cell detachment agent in the cell culture medium that is supplied to the cell culture chamber 12 via the pump 28c.
  • the decreased concentration of cell detachment agent will decrease the rate at which cells are detached from the surface, thereby increasing the cell density or confluency.
  • the cell detachment agent is supplied continuously from the supply of cell detachment agent 16 to the pre-reactor 14.
  • the controller 26 acts on the pump 28b to increase or decrease the rate at which the cell detachment agent is supplied to the pre-reactor 14.
  • the cell detachment agent is supplied semi-continuously from the supply of cell detachment agent 16 to the pre-reactor 14.
  • the controller 26 acts on the pump 28b to supply a quantity of cell detachment agent to the prereactor 14 at desired time intervals and/or in response to feedback from the sensor 24 in order to maintain a desired concentration of cell detachment agent in the cell culture medium.
  • the cell detachment agent may be a metalloprotease that enables adherent cells to be detached from a support er other adherent cells (e.g. in a cell aggregate).
  • Appropriate metal loproteases for use in the present invention are known in the art.
  • the metal loprotease may be collagenase.
  • the collagenase may be a collagenase selected from the group consisting of: collagenase type I, collagenase type II, collagenase type III, collagenase type IV, collagenase type V, collagenase type VI, and collagenase type VII.
  • collagenase type I to type VII are enzyme compositions comprising collagenase at increasing levels of purity, with collagenase type VII being pure collagenase.
  • the collagenase may be collagenase type I or collagenase type VII.
  • the metal loprotease may be a dispase.
  • the dispase may be dispase I or dispase II.
  • the cysteine protease may be of the CA clan.
  • the cysteine protease of the CA clan may be selected from the group consisting of ficin, papain, bromelain, cathepsin K and calpain. More suitably, the cysteine protease of the CA clan may be ficin and/or papain.
  • any suitable protease may be used in the present invention.
  • the protease is compatible with a cell culture media.
  • the protease is capable of detaching cells without substantially affecting cell growth or viability.
  • the present invention is based upon the surprising finding that aggregation can be reduced in a cell cluster based cell culture system by reducing overgrowth of cells and/or by preventing bridging between cell clusters in the cell culture medium.
  • the invention is based upon providing in the cell culture medium, suitably during the growth phase, a metalloprotease (such as a collagenase and/or dispase) and/or a cysteine protease (such as papain and/or ficin) which disrupts cell-cell interactions between different cell clusters within the cell culture and/or detaches a proportion cells from the cell cluster to prevent overgrowth.
  • a metalloprotease such as a collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • the method of the invention also provides a means for controlling the confluency of cells on an artificial support such as a microcarrier. As a result, the cells can be prevented from reaching high confluency and/or overgrowth.
  • the invention therefore provides a new means for reducing or indeed preventing aggregation (clumping) during cell culture.
  • serine proteases for example trypsin
  • metalloproteases and cysteine proteases having a different mechanism of action as compared to serine proteases.
  • serine proteases may transmit intracellular signals
  • metalloproteases and cysteine proteases act on the extracellular matrix, not with/on the cells and do not directly trigger any intracellular response.
  • the presence of a metalloprotease and/or cysteine protease in the cell culture medium reduces aggregation of cell clusters (such as aggregation of cell-populated microcarriers, spheroids, organoids, tissues, as appropriate).
  • the proportion of cell clusters which aggregate together may suitably be 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the total number of cell clusters in the cell culture medium.
  • the presence of aggregates in a cell culture medium may be substantially 0%, meaning that the protease essentially prevents aggregation of cell clusters.
  • a desired proportion of the cell clusters will remain in suspension in the cell culture medium, and will not become attached to other cell clusters present in the medium.
  • the presence of a metalloprotease and/or a cysteine protease in the culture medium also serves to reduce or prevent binding or aggregation of the cell clusters on a surface of a cell culture system, such as a bioreactor.
  • the present invention provides for a cell culture in a bioreactor where the cell clusters are maintained as a homogenous (suspended) population in the cell culture medium.
  • the method of the invention has the advantage of increasing the growth capacity of the cell culture, by reducing or preventing aggregation which may limit access of cells to nutrients and/or gases and result in a decrease or limitation of growth.
  • the method of the invention also improves harvesting of cells from the microcarriers, by avoiding overgrowth, cell clumping, reducing shear, and/or loss of viable cells due to aggregation.
  • a protease may be pure, or may contain trace amounts (e.g. 15% or less, or 10% or less) of other proteins or enzymes.
  • the collagenase type I may contain other enzymes such as caseinase, clostripain and trypsin.
  • the collagenase type I may have more than 125U/mg of collagenase; more than 200U/mg of caseinase; less than 4 ll/rng of clostripain and less than 0.5U/mg of tryptic activity.
  • protease is a collagenase
  • it may be that which is obtained from Clostridia histolyticum and is also used in human medicine. However, it can also be isolated from other Clostridia bacteria or tissues (e.g. Merck Index, No. 2477).
  • a suitable collagenase may be GibcoTM Collagenase Type I (product code 11500536 from Fisher Scientific) that is isolated from Clostridium histolyticum which contains average levels of collagenase, caseinase, clostripain and tryptic activities.
  • GibcoTM Collagenase Type I product code 11500536 from Fisher Scientific
  • Clostridium histolyticum which contains average levels of collagenase, caseinase, clostripain and tryptic activities.
  • FDA approved collagenases are known and available in the art. These include, for example, Santyl (Smith and Nephew); cellulite (Qwo, Endo International), and Xiaflex (Endo International).
  • a single metal loprotease i.e. collagenase or dispase
  • cysteine protease i.e. papain or ficin
  • a combination of two or more metal loproteases or cysteine proteases may be provided in a cell culture, in a single cell culture period.
  • one metalloprotease i.e. collagenase or dispase
  • one cysteine protease i.e.
  • papain or ficin may be used in cell culture in a single cell culture period, or combination of two or more metalloproteases (such as collagenase and dispase) or cysteine protease (i.e. papain and ficin) may be used in cell culture in a single cell culture period. Combinations of metalloproteases and cysteine proteases may also be used in cell culture in a single cell culture period.
  • metalloproteases such as collagenase and dispase
  • cysteine protease i.e. papain and ficin
  • the cell detachment agent may be provided to the cell growth chamber at a concentration which allows for the ratio of the rate of detachment of cells (e.g. detachment from a support or detachment from other adherent cells such as detachment from a cell aggregate) to the rate of growth of the cells to be in the range of 0.3:1 to 1 :3.
  • the cell detachment agent may be provided to the cell growth chamber at a concentration which maintains the cell culture at about 50% to 100% confluency.
  • Additional metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • Supplemental metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • supplemental metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • Supplemental metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • Regular intervals may, for example, be every hour, 2 hours, 3 hours, 4 hours, 5 hours 6, hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 36 hours, 48 hours or 72 hours.
  • Regular administration of additional metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • supplemental metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • these may depend upon factors such as the growth rate of the cells, the rate of detachment, the degree of confluency on the microcarriers, and/or the concentration of metalloprotease (such as collagenase and/or dispase) and/or cysteine protease (such as papain and/or ficin) in the cell culture media.
  • Supplemental metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • Supplemental metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • Supplemental metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • cell culture media may be added with cell culture media.
  • an amount of the existing cell culture media may be removed, to maintain the desired concentration of the protease (such as collagenase and/or dispase).
  • removal or addition of cell culture media does not amount to washing of the cells, and does not alter the growth phase of the cells when in the log phase. In a cell culture period, the cell culture media is not wholly removed, and wash media is not added to the cell culture.
  • the cell population may be cultured on a support in the cell culture medium, wherein the cell detachment agent continuously detaches a proportion of the cells in the cell population from the support.
  • the cell population may be a cell aggregate, wherein the cell detachment agent continuously detaches a proportion of the cells from the cell aggregate.
  • the metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • “continuously detaches a proportion of the cells” refers to continually removing a proportion of the adherent cells in the cell population (e.g. removing a proportion of the adherent cell population on a support, or removing a proportion of the adherent cell population in an aggregate). The detachment therefore occurs gradually over time and is not one discrete (bulk detachment) event.
  • the cell detachment agent e.g. a metalloprotease (such as collagenase and/or dispase) and/or a cysteine protease (such as papain and/or ficin)
  • a metalloprotease such as collagenase and/or dispase
  • a cysteine protease such as papain and/or ficin
  • the cell detachment agent continuously detaches a proportion of the cells in the cell population over the culture period.
  • the cell detachment agent e.g.
  • a metalloprotease such as collagenase and/or dispase
  • a cysteine protease such as papain and/or ficin
  • the continuous detachment therefore may serve to maintain the cell population at a confluency or density that enables the cell population to remain in log growth phase over the culture period (or at least avoid the stationary and lag phases of cell growth). This may be particularly useful in the context of continuous bioprocessing systems such a bioreactors.
  • the methods of the invention therefore typically maintain the adherent cell population at a confluency of between 40% and 95% (e.g. when adherent on a support). Optimal confluence is discussed elsewhere herein and would be readily determined by a person of skill in the art.
  • the cell detachment agent e.g. a metal loprotease (such as collagenase and/or dispase) and/or a cysteine protease (such as papain and/or ficin)
  • a proportion e.g. at least 5%, but no more than 60%
  • the cell detachment agent e.g. a metal loprotease (such as collagenase and/or dispase) and/or a cysteine protease (such as papain and/or ficin)
  • a proportion e.g. at least 0.5%, but no more than 7% per hour
  • Continuous detachment may maintain the cell confluency or density of the adherent cell population over the culture period (in other words, the cell confluency or density may in some examples increase or decrease by no more than 40% over the culture period).
  • Continuous detachment may maintain the cell confluency or density of the adherent cell population over the culture period (in other words, the cell confluency or density may in some examples increase or decrease by no more than 40% on weekly average for the culture period).
  • continuous detaching does not encompass bulk detachment (which is a term that is well known in the art).
  • bulk detachment may be referred to as the detachment of at least 60% of cells in the adherent cell population in 30 minutes at 37°C.
  • the rate of detachment may exceed the rate of proliferation.
  • the cell detachment agent e.g. a metalloprotease (such as collagenase and/or dispase) and/or a cysteine protease (such as papain and/or ficin)
  • the cell detachment agent may be provided in the cell culture medium at a concentration which allows for the ratio of the rate of detachment of cells (e.g. from the support or from other adherent cells e.g. from a cell aggregate) to the rate of growth of the cells to be in the range of 0.3:1 to 1 :3.
  • the rate of detachment may be a third or more of the rate of proliferation.
  • the rate of proliferation may be up to 3 times the rate of detachment.
  • the rate of detachment and rate of proliferation may be determined by, e.g. imaging, bioreactor sensors/probes, analytical methods, all of which are well known in the art.
  • the detached cells, or a proportion of the detached cells may be harvested at least once during the cell culture period.
  • the detached cells, or a proportion of the detached cells may be harvested more than once during the cell culture period.
  • Harvesting of detached cells may take place every hour, 2 hours, 3 hours, 4 hours, 5 hours 6, hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 36 hours, 48 hours or 72 hours.
  • Suitable methods of removing detached cells from the culture include using deadend or crossflow/tangential fluid flow filtration, a centrifuge, acoustic separation or a microfluidic based system.
  • detached cells are not harvested, and may remain in the cell culture until after the cell culture period.
  • the cells Before or after step i) of a method of the invention, the cells may be passaged one or more times.
  • a method may comprise removing cells from a cell culture and placing said cells onto e.g. a new microcarrier.
  • the cells are a population of cells which are detached by a method of the invention.
  • a method of the invention may additionally comprise monitoring or determining the viability of the cells in the cell culture.
  • Cell viability may be measured through measurement of cell proliferation or metabolic activity. Other methods include flow cytometry and immunohistochemistry.
  • the metalloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • a substantial detrimental effect may be considered as a 10% decrease in cell viability over the culture period compared to the cell viability observed in the absence of the metalloprotease (such as collagenase and/or dispase) and/or cysteine protease (such as papain and/or ficin).
  • a method of the invention may additionally comprise monitoring or determining the confluency of cells (e.g. on the support).
  • Cell confluency may be measured using chemical dyes (e.g. thymidine, Alamar blue, XTT or others available in the art), qualitative visual measurement status, or using an image processing method including for example an Olympus CKX53 culture microscope, CKX-CCSW confluency checker software and the Air Fraction output.
  • chemical dyes e.g. thymidine, Alamar blue, XTT or others available in the art
  • qualitative visual measurement status e.g. thymidine, Alamar blue, XTT or others available in the art
  • an image processing method including for example an Olympus CKX53 culture microscope, CKX-CCSW confluency checker software and the Air Fraction output.
  • cells may also be imaged and analysed using Cytation 1 , loLight, Jiusion USB digital Microscope and Imaged.
  • additional protease for example collagenase and/or dispase
  • Account may be taken of the overall concentration of protease (for example collagenase and/or dispase), to remain within optimal limits for the cell type.
  • Cell confluency may also be referred to as the density of adhered cells (e.g. on the support or the density of cells in the cell cluster).
  • Cell density may be determined using e.g. microscopy, acoustic resonance densitometry, laser induced fluorescence, fluorescence microscopy, capacitance impedance, turbidity, a biomass permittivity probe, a Raman probe and a cell counter e.g. CCD imaging using Trypan blue.
  • Cell density may also be used to determine the rate of detachment of cells from a cell cluster, as described elsewhere herein.
  • a method of the invention may include monitoring or determining the growth phase of the cells in culture. This may be performed using visual means, such as checking cell shape. Any suitable cell imaging method may be used, for example imaging or otherwise analyzing cells, for example fluorimeters, luminometers, cameras, microscopes, plate readers, cell analyzers, and confocal imaging systems.
  • the culture period does not comprise a step of washing the cells.
  • a washing step may be included. Washing may be performed using any suitable method dependent on the choice of cell (and e.g. the choice of cell cluster). Washing may include aspirating cell culture media, and be performed by placing the cells in a physiological buffer.
  • a method of the invention may include counting the cells.
  • substantially all the cells of the cell population do not differentiate. Therefore, substantially all of the cells of the cell population will not show signs of differentiation, for example differentiation markers or changes in cell shape or morphology.
  • a method of the present invention may further comprise culturing the cells under conditions suitable to allow differentiation.
  • a method of the present invention may further comprise cryopreservation of harvested cells.
  • Any suitable source of an exogenous metal loprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • the metal loprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • the metal loprotease is compatible with a cell culture media.
  • the metal loprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • any suitable concentration or amount of cell detachment agent may be used provided that it is capable of detaching the requirement amount of cells (e.g. to provide continuous detachment a proportion of the cells in the cell population during the culture period).
  • metal loprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • suitable concentrations and amounts based on the disclosure herein and their common general knowledge. The exact amount to be used may depend on a number of factors e.g. the desired rate of detachment, the cell culture medium, whether static or dynamic conditions are used etc. Some non-limiting examples are provided herein.
  • a suitable concentration may therefore be determined by a person of skill in the art.
  • collagenase it may be provided in a cell culture at or below 500U/ml.
  • it may be provided in a cell culture at or below 215U/ml.
  • the collagenase may be provided in a cell culture at or below 120U/ml.
  • the collagenase may be provided in a cell culture at or below 110U/ml or at or below 50U/ml.
  • the collagenase is collagenase type I
  • it may be provided in a cell culture at or below 215U/ml.
  • the collagenase type I may be provided in a cell culture at or below 120U/ml.
  • the collagenase type I may be provided in the cell culture at or below 110U/ml.
  • the collagenase type may be provided in the cell culture at or below 80U/ml.
  • the collagenase type may be provided in the cell culture at or below 50U/ml.
  • the collagenase type I may be provided in the cell culture at or below 20U/ml.
  • the collagenase type I may be provided in the cell culture at or below 10U/ml.
  • the collagenase type I may be provided in the cell culture at or below 5U/ml.
  • the collagenase type I may be provided in the cell culture at or below
  • the collagenase type I may be provided in the cell culture at or below 2U/ml.
  • the collagenase type I may be provided in the cell culture at or below 1 U/ml or any range formed from any of the upper or lower limits of the aforementioned ranges, or any integer therebetween, or at least, no more than, below or above any value of an aforementioned ranges.
  • the lower end of the range may be 0.005 U/ml e.g. 0.008 ll/ml.
  • the collagenase is collagenase type I, it may be provided in a cell culture at a concentration between 0.008U/ml and 20U/ml.
  • the collagenase is collagenase type I
  • it may be provided in a cell culture at a concentration between 5U/ml and 20U/ml, or at a concentration between 9U/ml and 20 U/ml.
  • the collagenase is collagenase type I
  • it may be provided in a cell culture at a concentration between 0.008U/ml and 10U/ml.
  • the collagenase is collagenase type I
  • it may be provided in a cell culture at a concentration between 5U/ml and 10U/ml, or at a concentration between 0.008U/ml and 5 U/ml.
  • the collagenase is collagenase type VII
  • it may be provided in a cell culture at or below 500U/ml.
  • the collagenase type VII is provided in a cell culture at or below 300U/ml.
  • the collagenase type VII is provided in a cell culture at or below 200U/ml.
  • the collagenase type VII is provided in a cell culture at or below 150U/ml.
  • the collagenase type VII may be provided in a cell culture at or below 120U/ml.
  • the collagenase type VII may be provided in the cell culture at or below 110U/ml.
  • the lower end of the range may be 50 U/ml.
  • the collagenase is collagenase type VII
  • it may be provided in a cell culture at a concentration between 50 U/ml and 300U/ml.
  • the collagenase is collagenase type VII
  • it may be provided in a cell culture at a concentration between 50U/ml and 200U/ml, or at a concentration between 50U/ml and 150 U/ml.
  • the collagenase is collagenase type VII
  • it may be provided in a cell culture at a concentration between 50 U/ml and 120U/ml.
  • the collagenase is collagenase type VII
  • it may be provided in a cell culture at a concentration between 50 U/ml and 110U/ml.
  • the enzyme is dispase
  • it may be provided in the cell culture medium at or below 13U/ml.
  • the dispase is present in the cell culture medium at or below 6.5U/ml.
  • the dispase is present in the cell culture medium at or below 3U/ml.
  • the dispase is present in the cell culture medium at or below 2U/ml or at or below 1 U/ml.
  • the dispase is present in the cell culture medium at or below 0.8U/ml or at or below 0.5U/ml. In each of these cases the lower end of the range may be 0.0005 U/ml e.g. 0.0008 U/ml.
  • the dispase may be provided in a cell culture at a concentration between 0.0008U/ml and 3U/ml.
  • the dispase may be provided in a cell culture at a concentration between 0.0008U/ml and 2U/ml, or at a concentration between 0.0008 U/ml and 1 U/ml.
  • the dispase may be provided in a cell culture at a concentration between 0.0008 ll/ml and 0.8U/ml.
  • the dispase may be provided in a cell culture at a concentration between 0.0008 ll/ml and 0.5 ll/ml.
  • An enzyme unit (II) is a measure of an enzyme’s catalytic activity, and an enzyme unit (II) is the amount of enzyme which catalyses the conversion of one micromole of substrate per minute under the specified conditions.
  • the enzyme activity may be expressed in kalals (the enzyme activity which converts one mole of substrate per second under the specified conditions).
  • the enzyme activity unit may be a Collagenase Degrading Units (CDU or Mandi) where one CDU catalyses the hydrolysis of one micromole of L-leucine equivalents from collagen in 5 hours at 37°C, pH 7.4 (Lockhardt et al J. Stem Cell Res Ther, 5:321 (2015)).
  • the amount of enzyme as a measure of weight/volume (e.g. mg/ml) in the cell culture can be determined, for example by using the % enzyme and units of enzyme activity provided by the distributor.
  • the skilled person can determine the amount of product required to provide the desired enzyme units.
  • the enzyme units of 110U/ml is equivalent to 0.5mg/ml based on the proportion of protease in the product, and the weight of product used in the cell culture.
  • the enzyme units of 215U/ml for collagenase type I as used herein is equivalent to 1 mg/ml.
  • enzyme units of 6.5U/ml is equivalent to 0.5mg/ml and enzyme units of 13U/ml is equivalent to 1 mg/ml.
  • a metal loprotease e.g. collagenase and/or dispase
  • a metal loprotease may be provided in the cell culture media at or below 1 mg/ml, or suitably at or below 0.5mg/ml.
  • a metal loprotease e.g. collagenase and/or dispase
  • collagenase and/or dispase may be provided at or below about 1 mg/ml, 0.9mg/ml, 0.8mg/ml, 0.7mg/ml, 0.6mg/ml, 0.5mg/ml, 0.4mg/ml, 0.3mg/ml, 0.2mg/ml, 0.1mg/ml, more suitably at or below 0.09mg/ml, 0.08mg/ml, 0.07mg/ml, 0.06mg/ml, 0.05mg/ml, 0.04mg/ml, 0.03mg/ml, 0.02mg/ml, 0.01 mg/ml, 0.005mg/ml, 0.001 mg/ml, or 0.0005mg/ml.
  • the metal loprotease e.g. collagenase and/or dispase
  • the metal loprotease may be provided at a concentration in the cell culture media of about 0.0001 to 1mg/ml, 0.001 to 0.9mg/ml, 0.01 to 0.9mg/ml, 0.1 to 0.9mg/ml, or any range formed from any of the upper or lower limits of the aforementioned ranges, or any integer therebetween, or at least, no more than, below or above any value of an aforementioned ranges.
  • the exogenous cysteine protease may be provided at or below about 1 mg/ml, 0.9mg/ml, 0.8mg/ml, 0.7mg/ml, 0.6mg/ml, 0.5mg/ml, 0.4mg/ml, 0.3mg/ml, 0.2mg/ml, 0.1 mg/ml, more suitably at or below 0.09mg/ml, 0.08mg/ml, 0.07mg/ml, 0.06mg/ml, 0.05mg/ml, 0.04mg/ml, 0.03mg/ml, 0.02mg/ml, 0.01mg/ml, 0.005mg/ml, 0.001 mg/ml, or 0.0005mg/ml.
  • the exogenous cysteine protease e.g. papain and/or ficin
  • ficin may be provided at a concentration in the cell culture media of about 0.0001 to 1 mg/ml, 0.001 to 0.75mg/ml, 0.001 to 0.5mg/ml, 0.01 to 0.25mg/ml, or any range formed from any of the upper or lower limits of the aforementioned ranges, or any integer therebetween, or at least, no more than, below or above any value of an aforementioned ranges.
  • ficin may be provided at a concentration in the cell culture media of about from 0.005 to 0.1 mg/ml, for example of about from 0.01 to about 0.05 mg/ml.
  • ficin may be provided at a concentration in the cell culture media of about from 0.0001 to about 0.1 BAPA U/rnL, for example from about 0.0005 to about 0.01 , from about 0.001 to about 0.009, or for example from about 0.002 to about 0.005 BAPA U/rnL.
  • ficin may be provided at a concentration of about 0.0033 BAPA U/rnL in the cell culture media.
  • papain may be provided at a concentration in the cell culture media of about 0.0001 to 1 mg/ml, 0.001 to 0.75mg/ml, 0.001 to 0.5mg/ml, 0.005 to 0.25mg/ml, or any range formed from any of the upper or lower limits of the aforementioned ranges, or any integer therebetween, or at least, no more than, below or above any value of an aforementioned ranges.
  • papain may be provided at a concentration in the cell culture media of about from 0.0001 to 0.07 mg/ml, for example of about from 0.001 to about 0.025 mg/ml.
  • papain may be provided at a concentration in the cell culture media of from about 0.001 to about 10 Til U/rnL, for example from about 0.05 to about 5, from about 0.1 to about 2 or for example from about 0.5 to about 1 Til U/rnL.
  • papain may be provided at a concentration of about 0.79 Til U/rnL in the cell culture media.
  • a population of cells is obtained in a way that would be understood by the skilled person, for example by obtaining a suitable tissue, and isolating the cells therefrom.
  • the cells may be one or more of cultured, passaged and separated, for example in a cell culture medium (without a cell detachment agent), prior to being seeded onto a support (which has been sterilized).
  • the population of cells may be obtained via a continuous bioprocess using a bioprocessing system according to the present invention.
  • the population of cells used for seeding may, therefore, be n-1 and/or n-2 stage cells.
  • the population of cells are seeded onto sterilized supports within the cell growth chamber 12.
  • the cell growth chamber 12 is supplied with a cell culture medium including the cell detachment agent (protease) via the pre-reactor 14 (Step 101).
  • the protease acts to continuously detach a proportion of the cells in the cell population from the support.
  • Cell culture medium containing the detached cells is collected in the collection container 20.
  • the control loop 22 is used to ensure that the rate at which cells are detached from the support matches the rate of cell growth. In this way, the cell layer does not become too densely populated, or over-confluent.
  • the control loop 22 enables the cell layer to be maintained at a predetermined target cell density, for example 80 to 100% confluency or 60,000 cells/cm 2 , during the cell culture period.
  • the detachment of cells therefore occurs gradually over time and is not one discrete (bulk detachment) event, for example a bulk detachment event at the end of a culture period.
  • the sensor 24 measures the cell density (Step 102).
  • the controller 26 receives the measured cell density from the sensor 24 and compares the measured cell density with the predetermined target cell density (Step 103).
  • the controller 26 acts to decrease the concentration of cell detachment agent within the cell culture medium (Step 105). This may be achieved by decreasing the activity of the pump 28b in order to reduce the rate at which the cell detachment agent is supplied from the cell detachment agent reservoir 16 to the pre-reactor 14.
  • the controller 26 acts to increase the concentration of cell detachment agent within the cell culture medium (Step 105). This may be achieved by increasing the activity of the pump 28b in order to increase the rate at which the cell detachment agent is supplied from the cell detachment agent reservoir 16 to the pre-reactor 14.
  • Step 104b In the event the measured cell density matches the predetermined target cell density (Step 104b), no action is required from the controller 26 and the activity of the pumps 28a, 28b, 28c may be maintained at the current levels so as to maintain the concentration of the cell detachment agent within the cell culture medium.
  • action is taken by the controller 26 in order to increase or decrease the concentration of the cell detachment agent relative to the target concentration, a time lag may be applied by the controller before further action is taken to modify the concentration of cell detachment agent within the system.
  • the controller may, for example, be programmed to wait a period of time, for example 4 to 6 hours, before repeating step S105.
  • cells may be detached continuously, meaning that under the method of the invention, the stationary phase of growth is not reached as the metalloprotease (for example collagenase and/or dispase) and/or cysteine protease (such as papain and/or ficin) present in the cell media continually detaches the cells from the cell clusters in the cell culture medium.
  • metalloprotease for example collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • the method of the invention is associated with a number of advantages.
  • the method enables an increase in the yield of cells from the cell culture, by maintaining the cells in the log phase of growth for a longer period of time.
  • the method provides advantages of reducing the amount of resources required to produce a desired yield, and consequently the resource or carbon footprint.
  • one sensor 18 is used to measure the density of adherent cells within the cell growth chamber. It will be understood that, in alternative embodiments of the invention more than one sensor may be provided to measure the density of adherent cells within the cell growth chamber. Each sensor of the at least one sensor may measure a different parameter or characteristic that is indicative of cell density. One sensor may, for example, measure the cell density using a direct method, e.g. a microscopic method, and another sensor may, for example, measure the cell density using an indirect method, e.g. by measuring ATP production within the cell growth chamber.
  • a direct method e.g. a microscopic method
  • an indirect method e.g. by measuring ATP production within the cell growth chamber.
  • control loop 22 measures and regulates the density of adherent cells in the cell grown chamber 12.
  • the continuous bioprocessing system includes a second control loop 30.
  • the second control loop 30 will be described with reference to Figure 2, which illustrates a continuous bioprocessing system including a first control loop 22 (as described with reference to Figures 1 and 6) and a second control loop 30.
  • the second control loop 30 is for measuring and regulating the continuous bioprocessing system based on an assessment of the cell culture medium in which the non-adherent or detached cells are suspended.
  • the second control loop includes a sensor 32, which is configured to measure (e.g. quantify) the amount of a nutrient or other component of the cell culture medium.
  • the sensor 32 may, for example be used to measure the amount of oxygen or glucose or ATP in the cell culture medium.
  • the controller 26 acts on the pump 28d to allow the supply of used medium from the collection container 20 to the pre-reactor 14.
  • the used medium from the collection container 20 is mixed with new, or un-used medium, from the reservoir 18 and cell detachment agent from the reservoir 16 and supplied to the cell culture chamber 12 via the pump 28c. Collected cells from the collection chamber 20 are transferred to the collection chamber 34, from where they can be harvested.
  • the controller 26 acts on the pump 28d to prevent the supply of used medium from the collection container 20 to the pre-reactor 14. In this instance, the used medium is transferred with the collected cells to the collection chamber 34, from which it can be discarded once the cells have been harvested.
  • the continuous bioprocessing system includes a control loop 36.
  • the further control loop 36 will be described with reference to Figure 3, which illustrates a continuous bioprocessing system including a first control loop 22 (as described with reference to Figures 1 and 6) and a further control loop 36.
  • the further control loop 36 is for measuring and regulating the continuous bioprocessing system based on an assessment of a characteristic, e.g. the quality, of the non-adherent or detached cells.
  • the further control loop includes a sensor 38, which is configured to determine a phenotype or other characteristic of the non-adherent or detached cells.
  • the sensor 38 may, for example be used to measure a marker of adherence in order to assess the cells physiological adhesion capacity after collection.
  • the marker may be a cell adhesion molecule (CAM), for example an integrin, a cadherin, a catenin or a syndecan.
  • CAM cell adhesion molecule
  • the controller 26 acts on the pumps 28a, 28b to increase or decrease the supply of new or un-used cell culture medium and/or cell detachment agent from the reservoirs 18, 16, respectively, to the pre-reactor 14. In this way, the quality of the harvested cells may be optimised.
  • the continuous bioprocessing system may include a first control loop 22 (as described with reference to Figures 1 and 6), a second control loop 30 (as described with reference to Figure 2) and a further control loop 36 (as described with reference to Figure 3).
  • a first control loop 22 as described with reference to Figures 1 and 6
  • a second control loop 30 as described with reference to Figure 2
  • a further control loop 36 as described with reference to Figure 3
  • An example of such a continuous bioprocessing system is illustrated in Figure 4.
  • Like reference numerals depict like features and so will not be described further.
  • the cell detachment agent and the cell culture medium are combined in a pre-reactor 14 before being supplied to the cell culture chamber 12. It will be understood that, in other examples, the cell detachment agent and the cell culture medium might be supplied directly to the cell culture chamber 112, as will now be described in connection with the bioprocessing system 110 with reference to Figure 7.
  • the continuous bioprocessing system 110 includes a cell culture chamber 112 (also referred to herein as a cell culture reactor or as a bioreactor), a first fluid reservoir 116 in the form of a supply of a cell detachment agent, e.g. an enzyme such as a protease, a second fluid reservoir 118 in the form of a supply of cell culture medium, a collection chamber 120 and a control loop 122.
  • the control loop 122 includes a sensor 124 and a controller 126.
  • the bioprocessing system 110 also includes a number of pumps (not shown).
  • the supply of cell culture medium 118 is in fluid communication with the cell culture chamber 112 such that fresh (i.e. new or unused) cell culture medium can be supplied directly to the cell culture chamber 112 via a pump (not shown).
  • the supply of cell detachment agent 116 is in fluid communication with the cell culture chamber 112 such that the cell detachment agent can be supplied directly to the cell culture chamber 112 via a pump (not shown).
  • the cell detachment agent (metalloprotease (such as collagenase and/or dispase) and/or cysteine protease (such as papain and/or ficin)) is supplied to the cell culture chamber 112 at a target concentration of cell detachment agent in order to ensure that, as the adherent cells grow and proliferate within the cell culture chamber 112, cells are continuously detached from the surface and collected, together with spent (or used) cell culture medium within the collection chamber 120.
  • metaloprotease such as collagenase and/or dispase
  • cysteine protease such as papain and/or ficin
  • the detachment of cells therefore occurs gradually over time and is not one discrete (bulk detachment) event, for example a bulk detachment event at the end of a culture period.
  • the control loop 122 is provided to ensure that the density or confluency of adherent cells within the cell culture chamber 112 is optimum for further cell growth and proliferation.
  • the sensor 124 is a biomass analysis sensor and is used to assess or determine the density or confluency of the adherent cells.
  • the biomass data in other words the measured or determined density or confluency of the adherent cells, is transmitted or otherwise relayed to the controller 126.
  • the controller 126 is a proportional-integral-derivative (or PID) controller.
  • the controller 126 continuously calculates an error value e(t) as the difference between the desired cell density or confluency and the measured cell density or confluency.
  • the controller 126 acts cell detachment agent reservoir 116 to adjust the quantity, concentration or activity of the cell detachment agent that is supplied to the cell culture chamber 112 from the second reservoir 116.
  • the controller 126 will act to increase the supply of cell detachment agent to the cell culture chamber 112.
  • the increased concentration of cell detachment agent will increase the rate at which cells are detached from the surface, thereby reducing the cell density or confluency within the cell culture chamber 112.
  • the controller 126 will act to decrease the supply of cell detachment agent to the cell culture chamber 112.
  • the decreased concentration of cell detachment agent will decrease the rate at which cells are detached from the surface, thereby increasing the cell density or confluency within the cell culture chamber 112.
  • the bioprocessing system 110 may include a second feedback, or control, loop 130.
  • the second feedback loop 130 includes a sensor 132 and is connected to the controller 126.
  • the second feedback loop 130 corresponds to the feedback loop 30 as described above.
  • cell culture medium is continuously transferred from the cell culture chamber 112 to the collection chamber 120 and that fresh and/or replenished cell culture medium is continuously transferred to the cell culture chamber 112 in order to ensure that the adherent cells within the cell culture chamber 112 have a continuous supply of the nutrients that are required for optimum growth and proliferation.
  • the supply of cell detachment agent to the cell culture medium may be continuous or may be semi-continuous (e.g. intermittent).
  • the cell detachment agent may be supplied continuously during the culture of the adherent cells.
  • the cell detachment agent may be supplied at specific time intervals during the culture of adherent cells.
  • the amount of the cell detachment agent supplied in either example may be increased or decreased in response to a measured cell density or confluency.
  • the cell detachment agent is supplied throughout the culture process (i.e. not just in a single discrete (bulk detachment) event, for example a harvesting event or step at the end of a culture period.
  • the bioprocessing system 110 may include a further feedback loop 136.
  • the further feedback loop 136 includes a sensor 138 and is connected to the controller 126.
  • the further feedback loop 136 corresponds to the feedback loop 36 as described above.
  • the bioprocessing system 110 may include either one of the second feedback loop 130 and the further feedback loop 136, or both or neither of the second feedback 130 and the further feedback loop 136.
  • the cells generated from the method of the invention may be used in a variety of research, diagnostic, drug screening, therapeutic, medical, industrial or food based applications.
  • a cell culture produced by a method of the present invention may find use in the cultured meat industry.
  • the adherent cells are grown on a surface or support within the cell culture chamber.
  • the cells may be adhered to other cells, for example as part of an aggregate of cells or as part of, or within, a tissue.
  • a cell detachment agent is used to continuously detach adherent cells.
  • the cell detachment agent described in detail is a protease, such as a collagenase or a dispase.
  • other cell detachment agents may also be used, for example the cells may adhere to a support by virtue of a coating on the support (e.g. a Cw- TPGPQGIAGQRGDS coating) that facilitates cell attachment.
  • a coating on the support e.g. a Cw- TPGPQGIAGQRGDS coating
  • cell detachment agent that interferes with the interaction between the cell and coating may be used to continuously detach the adherent cells from the coating.
  • Several appropriate coating and cell detachment agent combinations are known in the art.

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Abstract

La présente invention concerne un système de biotraitement de cellules adhérentes. Le système comprend : une chambre de croissance cellulaire ; un réservoir de fluide en communication avec la chambre de croissance cellulaire ; et une boucle de régulation pour mesurer et réguler la densité des cellules adhérentes dans la chambre de croissance cellulaire. La boucle de régulation comprend au moins un capteur conçu pour mesurer la densité des cellules adhérentes dans la chambre de croissance cellulaire ; et un dispositif de régulation conçu pour réguler automatiquement l'apport d'un agent de détachement cellulaire du réservoir de fluide à la chambre de croissance cellulaire en réponse à la densité mesurée des cellules adhérentes.
PCT/GB2023/053301 2022-12-20 2023-12-19 Système de biotraitement Ceased WO2024134176A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20140030805A1 (en) * 2011-04-15 2014-01-30 Pluristem Ltd. Methods and systems for harvesting cells
WO2015165700A1 (fr) * 2014-04-28 2015-11-05 Vivabiocell Spa Dispositif automatisé de culture et de récolte de cellules
US20170037421A1 (en) * 2014-04-28 2017-02-09 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Fermentation systems
US20220282203A1 (en) * 2021-03-07 2022-09-08 Cellino Biotech, Inc. Platforms and systems for automated cell culture

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140030805A1 (en) * 2011-04-15 2014-01-30 Pluristem Ltd. Methods and systems for harvesting cells
WO2015165700A1 (fr) * 2014-04-28 2015-11-05 Vivabiocell Spa Dispositif automatisé de culture et de récolte de cellules
US20170037421A1 (en) * 2014-04-28 2017-02-09 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Fermentation systems
US20220282203A1 (en) * 2021-03-07 2022-09-08 Cellino Biotech, Inc. Platforms and systems for automated cell culture

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Title
LOCKHARDT ET AL., J. STEM CELL RES THER, vol. 5, 2015, pages 321
MIOTTO ET AL.: "Developing a Continuous Bioprocessing Approach to Stromal Manufacture", ACS APPLIED MATERIALS & INTERFACES, vol. 9, no. 47, 2017, pages 41131 - 41142

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