Cell culturing of adherent cells

Abstract

This invention relates to methods for the continuous culture of adherent cells.

Description

[0001]CELL CULTURING OF ADHERENT CELLS This invention relates to methods for the continuous culture of adherent cells. BACKGROUND It is well-known that regenerative medicine, and in particular cell-therapy techniques, has been expanding its repertoire in terms of both purposeful cell types and applications, raising the potential to cure a wide range of diseases. However, the high number of cells required for some treatments (this can be up to 1 billion) presents, in terms of cell manufacture, a major challenge. Furthermore, as the final product is often represented by the cells themselves, numerous requirements have to be met under the current regulatory framework for cellular therapy products. Thus, implementing and adapting industrial processes, previously developed for large-scale mammalian cell culture for the production of biologics, has been attempted in order to sustain such a great demand for cells and to meet the required conditions. While substantial increases in cell yield have been achieved, some other critical production steps are still undergoing optimization. For instance, improved cell harvesting methods for easier, more effective, and less invasive recovery of the expanded cells from their culture substrates are required. When adherent cells are grown in culture, 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. When 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. In 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. It is an object of the invention to improve recovery of cells from a cell culture, and thereby ameliorate some of the problems associated with the prior art. BRIEF SUMMARY OF THE DISCLOSURE In one aspect of the present invention, there is provided a method of cell culture of an adherent cell population, the method comprising: (i) culturing the cell population in a cell culture medium comprising a metalloprotease and / or cysteine protease for a culture period which comprises all or part of the log phase of growth of the cell population, wherein the metalloprotease and / or cysteine protease continuously detaches a proportion of the cells in the cell population. In this context, it can be said that the cell culture medium is supplemented with the metalloprotease and / or cysteine protease. In other words, in one aspect of the present invention, there is provided a method of cell culture of an adherent cell population, the method comprising: (i) culturing the cell population in a cell culture medium comprising an exogenous metalloprotease and / or exogenous cysteine protease for a culture period which comprises all or part of the log phase of growth of the cell population, wherein exogenous metalloprotease and / or exogenous cysteine protease continuously detaches a proportion of the cells in the cell population. Suitably, the culture period may be at least 24 hours. Suitably, the metalloprotease may be selected from the group consisting of collagenase and / or dispase. Suitably, the collagenase and dispase may be exogenous. Suitably, the cysteine protease may be of the CA clan. Suitably, 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. In another aspect of the present invention, there is provided a method of cell culture of an adherent cell population, the method comprising: (i) culturing the cell population in a cell culture medium comprising a metalloprotease and / or cysteine protease for a culture period that is at least 24 hours, wherein the metalloprotease and / or cysteine protease continuously detaches a proportion of the cells in the cell population. In this context, it can be said that the cell culture medium is supplemented with the metalloprotease and / or cysteine protease. In other words, in another aspect of the present invention, there is provided a method of cell culture of an adherent cell population, the method comprising: (i) culturing the cell population in a cell culture medium comprising an exogenous metalloprotease and / or cysteine protease for a culture period that is at least 24 hours, wherein the metalloprotease and / or cysteine protease continuously detaches a proportion of the cells in the cell population. Suitably, in a method of the invention, the culture period comprises all or part of the log phase of growth of the cell population. Suitably, in a method of the invention, the cell culture medium may comprise a metalloprotease. Suitably, in a method of the invention, the cell culture medium may comprise collagenase. Suitably, in a method of the invention, the collagenase is 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. Notably, collagenase type I to type VII are enzyme compositions comprising collagenase at increasing levels of purity, with collagenase type VII being pure collagenase. Suitably, in a method of the invention, the collagenase is collagenase type I or collagenase type VII. These are the collagenases that are used herein to exemplify the invention, although the invention is not limited thereto. Suitably, in a method of the invention, the cell culture medium comprises a dispase. Suitably, in a method of the invention, the dispase is dispase I or dispase II. Suitably, in a method of the invention, the cell culture medium may comprise a cysteine protease. Suitably, the cysteine protease may be of the CA clan. Suitably, the cysteine protease of the CA clan may be selected from the group consisting of ficin, papain, bromelain, cathepsin K and calpain. More suitably, in a method of the invention, the cysteine protease of the CA clan may be ficin and / or papain. Suitably, in a method of the invention, the metalloprotease and / or cysteine protease is provided in the cell culture medium 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. Suitably, in a method of the invention, the metalloprotease and / or cysteine protease is provided in the cell culture medium at a concentration which maintains the cell culture at about 50% to 100% confluency. Suitably, in a method of the invention, the detached cells, or a proportion of the detached cells, may be harvested at least once during the cell culture period. Suitably, in a method of the invention, the detached cells, or a proportion of the detached cells, may be harvested more than once during the cell culture period. Suitably, in a method of the invention, the culture period does not comprise a step of washing the cells (e.g. it does not comprise the step of washing a support comprising the cells, or a step of washing a cell aggregate comprising the cells). Suitably, in a method of the invention, the cell culture medium is supplemented with metalloprotease and / or cysteine protease one or more times during the cell culture period. Suitably, in a method of the invention, the cell population is cultured on a support in the cell culture medium, wherein the metalloprotease and / or cysteine protease continuously detaches a proportion of the cells in the cell population from the support. Suitably, in a method of the invention, the support comprises a material selected from the group consisting of: plastic, polymer, glass, and metal. Suitably, in a method of the invention, 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. Suitably, in a method of the invention, the cell population is a cell aggregate, wherein the metalloprotease and / or cysteine protease continuously detaches a proportion of the cells from the cell aggregate. Suitably, in a method of the invention, the cell population comprises cells selected from the group consisting of: skin, muscle, cervical, breast, and prostate cells. Suitably, a method of the invention comprises culturing the cell population in a cell culture medium lacking the metalloprotease and / or cysteine protease before step (i). Suitably, a method of the invention comprises adjusting the metalloprotease and / or cysteine protease concentration and / or frequency of application to adjust the rate of cell detachment, wherein increasing the concentration and / or frequency of application increases the rate of continuous cell detachment and decreasing the concentration and / or frequency of application decreases the rate of continuous cell detachment. In a third aspect of the invention there is provided a cell culture vessel comprising a cell culture medium, an adherent cell population in the log phase of growth, and a metalloprotease and / or cysteine protease. Suitably, the cell culture vessel comprises a metalloprotease. Suitably, the metalloprotease is a collagenase and / or dispase. Suitably, the metalloprotease is a collagenase. Suitably, the collagenase is 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. Suitably, the collagenase is collagenase type I or collagenase type VII. Suitably, the metalloprotease is a dispase. Suitably, the dispase is dispase I or dispase II. Suitably, the cell culture vessel comprises a cysteine protease. Suitably, the cysteine protease may be of the CA clan. Suitably, 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. Suitably, the cell population is on a support in the cell culture medium. Suitably, the support comprises a material selected from the group consisting of: plastic, polymer, glass, and metal. Suitably, 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. Suitably, the cell population is a cell aggregate. Suitably, the cell population comprises cells selected from the group consisting of: skin, muscle, cervical, breast, and prostate cells. BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows that collagenase can successfully detach C2C12 cells in bulk similar to TrypLE (exemplified using collagenase type I). Graphs showing similar number of detached cells (A) and viability (B) using TrypLE and collagenase without altering cell adhesion (C) proliferation (D) and differentiation (E) potential (Scale bars 200µm). Figure 2 shows that collagenase is capable of detaching cells in a dose-dependent manner without altering the cell phenotype (exemplified using collagenase type I). Images showing attached (A) and detached (B) cells in the presence of varying concentrations of collagenase. The amounts of collagenase (U / mL) are shown in the Figure. The equivalent amounts in mg / mL for collagenase type I are: 2.635 U / mL (0.012 mg / mL); 3.500 U / mL (0.016 mg / mL); and 4.375 U / mL (0.020 mg / mL). (C) Graph showing increased cell detachment with increasing concentration of collagenase. (D) Representative images showing only expression of undifferentiated cell marker (Pax7) and not of differentiated cell marker (MHC) in attached cells at the end of the experiment (Scale bars 200µm). Figure 3 is a schematic showing the supplementation of collagenase as a strategy for continuous detachment of adherent cells in two systems: on flat and on microcarrier surfaces and in two conditions (static and dynamic). Exemplified using collagenase type I. Figure 4 shows the continuous detachment in a flat-static system. Cell detachment using 1.75 U / mL (equivalent to 0.008 mg / mL) of collagenase (collagenase type I) shows similar number of attached (A) and detached (B) cells over 3 weeks. Quantification showing similar number of attached (C) and detached (D) cells over time suggesting steady state of cell proliferation and cell yield (Scale bars 200µm). Figure 5 shows the continuous detachment in a flat-dynamic system. Exemplified using collagenase type I. (A) Images showing similar number of attached cells over a week in a flat dynamic system. (B) Quantification showing similar number of cells were harvest on day 7 compared to seeded cells on day 0. (C&D) Representative images showing expression of undifferentiated cell marker, Pax7 in both detached (C) and attached (D) cells (Scale bars 200µm). Figure 6 shows the continuous cell detachment from microcarrier surfaces when supplementing the media with collagenase (exemplified using collagenase type I, A). Stained cell nuclei showing microcarrier aggregation in controls (B) caused by cell overgrowth, while collagenase addition inhibits microcarrier bridging and cellular overgrowth (C). Quantification showing (D) average number of weekly detached cells using collagenase in the static system (over 27 days) and (E) total number of harvested cells from the microcarriers at the end of the experiment (Scale bars 200µm and 1000µm). Figure 7 shows attached cell behaviour. Cells detached from microcarriers using collagenase (A) can re-attach on flat tissue culture treated surface in serum plus media as well as the cells recovered from the microcarriers at the end of the experiment (B). (C) The collagenase-detached cells are also capable to attach on fresh microcarriers and stay in the continuous process in serum free media with collagenase. Exemplified using collagenase type I (Scale bars 200µm). Figure 8 shows continuous detachment in a microcarrier-dynamic system. Quantification showing daily (A) and weekly (B) number of cells detached using collagenase over 27 days in the dynamic system. (C) Representative images of nuclei stained cells on microcarriers and (D) final quantification showing seeded and harvested number of cells at the end of the experiment. Exemplified using collagenase type I (Scale bars 200µm and 1000µm). Figure 9 shows attached cell behaviour. Cells detached from microcarriers using collagenase (detached, A) can re-attach on flat tissue culture treated surface in serum plus media as well as the cells recovered from the microcarriers at the end of the experiment (attached, B). (C) Collagenase-detached cells can be cryopreserved and are capable to re- attach and grow on tissue culture treated plastic once defrosted (defrosted cryopreserved). Exemplified using collagenase type I (Scale bars 200µm). Figure 10 shows that collagenase can successfully detach fish skin fibroblasts in bulk. After 20h incubation of cells with collagenase are detached (A) and after collection (B) they are capable of reattaching (5h after seeding, C) and proliferating (48h after seeding, D). Exemplified using collagenase type I used at a concentration of 1505 U / mL (which is equivalent to 7 mg / mL, Scale bars 500µm). Figure 11 shows that dispase can successfully detach C2C12 cells continuously. Confluence of growing cells is maintained during the 3 days (attached, A) whilst cells are being detached (detached, B) at a comparable rate over time. Further experiments have also shown the same effect over 21 days (data not shown). Exemplified using dispase I used at a concentration of 0.0008 U / mL (which is equivalent to 0.00006 mg / mL or 60 ng / mL, Scale bars 200µm). Figure 12 shows that pure collagenase can successfully detach C2C12 cells continuously. Confluence of growing cells is maintained during the 3 days (attached) whilst cells are being detached (detached) at a comparable rate over time. Exemplified using collagenase type VII. (A) Collagenase VII used at a concentration of 106.35 Units / mL (equivalent to 0.07 mg / mL); (B) Collagenase VII used at a concentration of 53.18 Units / mL (equivalent to 0.035 mg / mL, Scale bars 200µm). Figure 13 shows Ficin and Performase® detach cells in a dose dependency manner. Images showing C2C12 cells growing on surfaces 6 days after incubation with different concentrations of Ficin (A) and Performase® (B) added to serum-free media (Scale bars 250μm). Figure 14 shows continuous cell detachment with Ficin (A) or Performase® (B). Images taken at day 0 and 10 showing the initial confluency (after cell seeding) and the confluency after continuous enzyme exposure, respectively. Images at day 10 show the confluency on the growth surface (attached) and the cells that detached during the last 3 days of the experiment (detached, Scale bars 250µm). Figure 15 shows Bulk detachment with Ficin or Performase®. C2C12 cells can be detached in bulk with Ficin (A) and Performase® (B) once added to the culture media. Cells detached already after 30 minutes with complete detachment occurring after 150 minutes (Scale bars 250µm). Figure 16 shows cell viability after Ficin or Performase® exposure. C2C12 cells detached in bulk with Ficin or Performase® maintained a high cell viability after 4h of exposure to high enzyme concentrations. DEFINITIONS As used herein, "about" will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value. The terms “comprise”, “comprises” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / or components of an aspect or embodiment of the invention, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The phrase “consisting essentially of” means that the scope of an aspect or embodiment is to be interpreted to encompass the specified materials or steps recited in the aspect or embodiment, including any materials or steps which do not materially affect the invention defined by the aspect or embodiment. Thus, the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.” 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. As used herein, “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. As used herein, “confluency” in the context of a support, refers to the percentage of the support which is covered by adherent cells. 50% confluency means that half of the support is covered with adherent cells. 100% confluency, or fully confluent, means that all of the support is covered with adherent cells. Over-confluency means that there is no available space on the support for new cells. In the context of a cell aggregate, cell density or aggregate size is more relevant. Means for determining cell density are described elsewhere herein. The term “cell culture medium” as used herein 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 support, and / or a cell culture medium, 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 as “exposing”, for example exposing cells to a cysteine protease shall be therefore understood as contacting cells with a cysteine protease. The term “adherent cells” as used herein 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 term “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). For example, the aggregate may comprise at least 10, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, or at least 109etc adjoining cells. In a preferred example, the adjoining cells are interconnected. As used herein, “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. Alternatively, 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. As described above, a matrix, substrate or scaffold may connect adjoining cells, to form an aggregate. The terms “matrix”, “substrate” and “scaffold” used interchangeably herein and are generically referred to as a “structure” within the aggregate. The structure may also facilitate or maintain aggregate formation. The structure may be naturally derived or synthetic. In one example, the structure may be a synthetic or natural polymer. Preferably, 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. In another example, the cells are adjoined via an extracellular matrix (ECM) in a manner that forms a multicellular aggregate. 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. Alternatively, the aggregates may be structure-free. Appropriate methods for cell culture with or without a structure are well known in the art. In a preferred example, the adjoining cells are interconnected. As used herein, “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). Examples of 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. Examples of suitable plant tissues or organs (that are obtained from a living plant) 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. Examples of 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. In one example, 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. In one particular example, 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. In some examples, 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. Accordingly, in one example, a multicellular aggregate comprises homogeneous or heterogeneous cell types. By “cell growth” it is meant the division, or proliferation, of the seeded cells. By “seeding” cells it is meant the application of an initial cell population to the cell culture media or support. “Seeded cells” are the cells initially applied to the support. The term “detach” or “detached” with reference to a cell on a support means separation of the cell from the support, so that it is no longer anchored or adhered to the support. The term “detach” or “detached” with reference to a cell aggregate means separation of the cell from the cell aggregate, so that it is no longer anchored or adhered to the cell aggregate. “Frequency of application” as used herein refers to the number of times a reagent, in particular the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin), is added to the cell culture medium, either prior to and / or during a cell culture period. “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. The term “metalloprotease” as used herein refers to a protease having one or more metal ions in the binding / active site. Examples of a metalloprotease include collagenase and / or dispase. The term "cysteine 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 are referred to herein as “thiol proteases” or “sulfhydryl proteases”. Many superfamilies of cysteine proteases are known in the art. Suitably, in the context of the present disclosure, 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. Suitably, 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. The term “CA clan” is based on the MEROPS classification scheme (https: / / www.ebi.ac.uk / merops / ). Suitably 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 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. DETAILED DESCRIPTION The present invention provides a method for adherent cell culture, in which the cells are detached (e.g. from a support or from each other e.g. from a cell aggregate) during the proliferation (or growth) phase, such that the stationary phase may be delayed, and cell yield may be increased. The invention is based upon providing in the cell culture medium 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 detaches the cells. The present invention therefore provides a method for continuous growth and detachment of adherent cells. In a defined period of cell culture which includes all or part of the log phase, the cells are maintained in both cell culture media and a metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin), under conditions suitable for cell growth / division. In a defined cell culture period, the cells may be detached continuously, meaning that under the method of the invention, the stationary phase of growth is not reached as the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) present in the cell media continually detaches the cells from the support and / or from other adherent cells e.g. from a cell aggregate). Surprisingly, the inventors found that other classes of proteases, such as serine proteases (for example trypsin), do not have the same utility in the context of continuous cell detachment. Without being wishing to be bound by this hypothesis, the inventors believe that is due to metalloproteases and cysteine proteases having a different mechanism of action as compared to serine proteases. Specifically, serine proteases may transmit intracellular signals, whereas metalloproteases and cysteine proteases act on the extracellular matrix, not with / on the cells and do not directly trigger any intracellular response. Continuous cell detachment may be especially useful in the context of a continuous bioprocessing system. In contrast to traditional cell culture systems, in which the cells are harvested in bulk after the cells enter the stationary phase, the continuous bioprocessing system allows the culturing and harvesting of adherent cells with minimal human intervention and increased productivity per unit volume. In traditional cell culture systems, an enzyme, such as trypsin, is added to the cell culture vessel at the end of the process, in a harvesting step, in order to detach the cells, and allow the cells to be harvested from the culture vessel. In contrast, in continuous bioprocessing the cell detachment agent (such as a metalloprotease and / or cysteine protease) may be supplied to the cell culture at all times (not just at the end of the process) to allow a small proportion of cells to detach, enabling other cells to divide. When a small proportion of cell detaches, detachment of cells therefore occurs gradually over time and is not one discrete (bulk detachment) event. It will be understood that in the context of the present disclosure, the supply of cell detachment agent (such as a metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin)) to the cell culture medium may be continuous or may be semi-continuous (e.g. intermittent). In some examples of the invention, the cell detachment agent may be supplied continuously during the culture of the adherent cells. In other examples of the invention, 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. In either example, 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 methods of the invention are associated with a number of advantages. For example, 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. In addition, the method provides advantages of reducing the amount of resources required to produce a desired yield, and consequently the resource or carbon footprint. In one aspect of the present invention, there is provided a method of cell culture of an adherent cell population, the method comprising: (i) culturing the cell population in a cell culture medium comprising a metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) for a culture period which comprises all or part of the log phase of growth of the cell population, wherein the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) continuously detaches a proportion of the cells in the cell population. In another aspect of the present invention, there is provided a method of cell culture of an adherent cell population, the method comprising: (i) culturing the cell population in a cell culture medium comprising a metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) for a culture period that is at least 24 hours, wherein the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) continuously detaches a proportion of the cells in the cell population. A method of the invention may comprise one or more steps prior to step i). A method may comprise for example, obtaining a cell population from a suitable source. The present invention is suitable for the growth of any adherent cell. Anchorage dependent cells are typically derived from a multi-cellular organism. Suitably, 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. In some embodiments, the cells are mammalian cells (e.g., human). The adherent cells may be non-mammalian cells, such as insect cells, bird cells, or fish cells. The cell may be a prokaryotic cell, for example a fungal cell or a bacterial cell. The adherent cells may be plant cells. Suitably, the adherent cells may be primary cells or immortalised cells. Merely by way of example, 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. Merely by way of example, 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. In one example, the cell population comprises cells selected from the group consisting of: skin, muscle, cervical, breast, and prostate cells. Suitable cells are described elsewhere herein, and the skilled person would understand how to obtain such a cell population. For example, the method may comprise obtaining a suitable tissue, and isolating the cells therefrom. The adherent cells are cultured as an adherent cell population. The adherent cell population may be in the cell culture medium as a cell aggregate (e.g. wherein the adherent cells adhere to each other rather than a support per se) or it may be cultured on a support. A cell population generated in the cell culture may form a layer of at least 50, 60, 70, 80, 90 or 100% confluency on a support. Thus, the cell layer may be referred to as being confluent. Suitably, the cell layer does not become over-confluent. Suitably, the cell layer is maintained at 80-100% confluency during the cell culture period. A layer of cells in contact with the support may be a monolayer of cells (meaning that the layer is one cell deep), or more than one layer of cells (two or more cells deep). The cells may comprise one type of cell, or two or more different cell types. 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. Before use, the support is typically sterilized. Sterilization may be performed, for example, by gamma-irradiation, by autoclave, by washing with alcohol or by ethylene oxide (ETO) gas treatment. Suitably, in the context of the present disclosure, the support may be any surface on which cells may be grown. Suitably, 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 coverslip, 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 macroanatomical 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. In one example, the support comprises a material selected from the group consisting of: plastic, polymer, glass, and metal. In one example, 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. It will be appreciated that all or part of a support may be made from or treated with a material that facilitates cell adhesion. A material that facilitates cell adhesion may be selected from the group consisting of a polyester, a polypropylene, a polyalkylene, a polyfluoro chloroethylene, 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. Suitably, the cellulose may be cellulose acetate. The extracellular matrix component may be one or more of fibronectin, vitronectin, chondronectin, or laminin. Suitably, the adherent material is electrostatically charged. Suitably, the adherent material may be coated with collagen or gelatin. Suitably, 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 20179 (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 non-adherent portion. The method of the present invention may be carried out in a growth chamber. As would be clear to a person of skill in the art, 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, air- lift, 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. Prior to step i), a method of the present invention may comprise one or more of culturing, maintaining, passaging, separating, and / or isolating the cell population. Where a cell is cultured or maintained in a cell culture medium prior to step i), this may be in the absence of metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin). Prior to step i) a suitable cell culture medium may be selected. 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. Suitably, a cell culture media is selected which does not substantially affect the activity of the selected metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin). Merely by way of example and not limitation, 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, MesencultTMBasal Medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), Ham's F-10, αMinimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium (IMDM), or any combination thereof. Other media that are commercially available (e.g., from Thermo Fisher Scientific, Waltham, MA) or that are otherwise known in the art can be equivalently used in the context of this disclosure. Again, only by way of example, 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, GLUTAMAX™ 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), AIM V media, Keratinocyte SFM, defined Keratinocyte SFM, STEMPRO® SFM, STEMPRO® complete methylcellulose medium, HepatoZYME- SFM, Neurobasal™ medium, Neurobasal-A medium, Hibernate™ A medium, Hibernate E medium, Endothelial SFM, Human Endothelial SFM, Hybridoma SFM, PFHM II, Sf 900 medium, Sf 900 II SFM, EXPRESS FIVE® medium, CHO-S-SFM, AMINOMAX-II complete medium, AMINOMAX-C100 complete medium, AMINOMAX-C140 basal medium, PUB-MAX™ karyotyping medium, KARYOMAX bone marrow karyotyping medium, and KNOCKOUT D-MEM, or any combination thereof. Suitably the cell culture medium may be serum-free. Suitably the cell culture medium may be glucose free. Alternatively, the cell culture medium may comprise serum. Appropriate types and amounts of serum are known e.g.1% FBS may be used. The metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be described as “exogenous metalloprotease and / or exogenous cysteine protease”. In this context, the term “exogenous” refers to a protease enzyme (specifically metalloprotease and / or cysteine protease) that is added to the cell culture media (i.e. the cell culture media has been supplemented with said enzymes). Accordingly, an “exogenous” refers to a protease enzyme (specifically metalloprotease and / or cysteine protease) is not expressed by the cells, or at least not expressed in the amounts present in the cell culture medium. In other word, the term “exogenous” refers to a protease enzyme (specifically metalloprotease and / or cysteine protease) from an external source (it is not naturally produced in this quantity by the cells in the cell culture medium). For example, term “exogenous collagenase” (or “exogenous dispase”) as used herein refers to a collagenase (or dispase) that is added to the cell culture media (i.e. the cell culture media has been supplemented with the collagenase (or dispase). It refers to a collagenase (or dispase) that has not been expressed by the cells that are present in the cell culture medium (metalloproteases expressed by the cells present in the cell culture would be considered as endogenous collagenase (or dispase) herein). It is therefore a collagenase (or dispase, or a cysteine protease such as papain and / or ficin) from an external source (it is not naturally produced in this quantity by the cells in the cell culture medium). For the avoidance of doubt, the cells in the cell culture media may be capable of producing these proteases as well, however, the collagenase (or dispase) referred to as exogenous herein are those that are added to the cell culture media from an external source. An exogenous metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may also be referred to as a cell culture media supplement or a supplemented metalloprotease (such as a collagenase and / or dispase) and / or a supplemented cysteine protease (such as papain and / or ficin) herein. Suitably, the cell culture medium may comprise one or more additional reagents, as required. These may be selected from, but are not limited to, the group consisting of an antibiotic, buffer, growth factor, hormone, nutritional supplement, indicator, and essential metals and minerals. In a next step prior to step i), the cells may be seeded on a support, or inoculated into the growth chamber (e.g. to grow as cell aggregates). Suitably, the cells are seeded onto the support after the support is incubated with the cell culture media or are inoculated directly into the cell culture media in the growth chamber. Methods of inoculating cells into cell culture media are well known and may be adapted according to the cell type. In addition, methods of cell seeding are known and available to the person skilled in the art and may be adapted according to the cell type and support. Two or more supports may be seeded simultaneously. The initial cell seeding density has to be efficient while allowing optimal cell proliferation within the support. The number of the cells to be seeded further depends on porosity of the scaffold material and any liquid absorption capability. For a porous support, the greater the porosity, the larger the number of cells that can be seeded. In some embodiments, the number of cells per g support (dry weight) is in the range of 2×106to 50×106cells. In addition, the porosity of the scaffold and the internal organization of support fibers contribute to the retention of the cells within and on the support. The method may comprise more than one sequential seeding steps on a support. The metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be provided in the cell culture medium, prior to the application of the cell culture medium to the cell culture. Therefore, the cell culture medium comprising a metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added to the support or to the growth chamber itself. Alternatively, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be provided separately to the cell culture medium. Suitably, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) is added to the cell culture prior to initiation of the culture period. Therefore metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added to the cell culture during the lag phase of cell growth. Alternatively, or additionally, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added to the cell culture towards the end of log phase of cell growth, or when the cells reach a confluency of at least 50%, at least 60%, at least 70% etc. In some examples, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added to the cell culture when the cells reach a confluency of at least 80% or 85%. Additional metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added to the cell culture during the culture period. Supplemental metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added to the cell culture once or more than once during a culture period. For example, supplemental metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added to the cell culture 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 times or more during a cell culture period. Supplemental metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added to the cell culture at regular or irregular intervals. 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 a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be referred to as “pulsing”. If supplemental metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) is added in at irregular intervals, these may depend upon factors such as the growth rate of the cells, the rate of detachment, the degree of confluency on the support, and / or the concentration of metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) in the cell culture media. Supplemental metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be provided to the cell culture where the rate of detachment is below the rate growth, for example by 10%, 20%, 30%, 40% or 50% or more. Supplemental metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added with cell culture media. In such an embodiment, an amount of the existing cell culture media may be removed, to maintain the desired concentration of the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin). Suitably, 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 culture is maintained under suitable cell culture conditions, at least for a cell culture period. Suitable cell culture conditions for different cell types will be known to a person skilled in the art. Typical growth conditions are 37˚C, at a relative humidity of 95% and CO2 availability of 5%. The pH may be maintained at 7-7.4. The cell culture may be maintained under static or dynamic conditions. In a dynamic system, movement is applied to the cell culture (for example by placing on a rocker) to allow movement of the cell culture media. In a static system, there is no mechanism provided for movement of the cell culture media. The cell culture period may be defined as comprising all or part of the log phase of growth of the cell population or may be defined as being at least 24 hours. The culture period may be at least 24 hours and may comprise all or part of the log phase of growth of the cell population. A log phase may be any period between the lag phase and the stationary phase, where the cells in the culture are actively proliferating. Suitably, the culture period includes all or part of the log phase, suitably at least 40%, 50%, 60%, 70%, 80% or at least 90% of the log phase. Suitably, the culture period does not include all or part of the lag phase or the stationary phase. The growth phase of the cell culture may be determined using any suitable method, for example visual or analytical methods (such as cell counting, DAPI staining etc). Suitably, a cell culture may be at least 24 hours (about 1 day), at least about 36 hours, at least about 48 hours (about 2 days), at least about 60 hours, at least about 72 hours (about 3 days), or more. Suitably, cell culture may be for at least about 84 hours, at least about 96 (about 4 days) hours, at least about 108 hours, at least about 120 hours (about 5 days), at least about 132 hours, at least about 144 hours (about 6 days), at least about 156 hours, at least about 168 hours (about 7 days), at least about 180 hours, at least about 192 hours (about 8 days), at least about 204 hours, at least about 216 hours (about 9 days), or more. Suitably, cell culture may be from about 1 day to about 9 days, for example from about 2 days to about 8 days, or from about 3 days to about 7 days. Suitably, a cell culture may be at least about 7 days, at least about 14 days, at least about 21 days, at least about 28 days, at least about 35 days, or more. Suitably, cell culture may be for at least about 40 days. Suitably, a cell culture may be at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, or more. Suitably, a cell culture may be at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, or more. In a method of the invention, suitably the cells (e.g. on the support) do not reach confluency during a cell culture period, or suitably do not become over-confluent. Suitably, a method of the present invention enables the cells to maintain a confluency of about 50-100%, more suitably 60-95%, more suitably 80-95%, or any range or integer therebetween, for example about 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% during a cell culture period. Suitably, the confluency is maintained below 100% during a cell culture period. The confluency may be maintained at a level as described herein by the use of the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) in the cell culture medium during the cell culture period, which allows for continuous detachment of the cells (e.g. from the support or from other adherent cells e.g. from a cell aggregate) as the cells as proliferating and the population is growing. In the methods of the invention, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) continuously detaches a proportion of the cells in the cell population. In this context, “continuously detaches a proportion of the cells in the cell population” 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. As would be clear to a person of skill in the art, continuous detachment occurs over a period of time (over the culture period, which is typically more than 24 hours). Accordingly, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) continuously detaches a proportion of the cells in the cell population over the culture period. Typically, the methods of the invention comprise detaching adherent cells from the cell population (using dispase and / or collagenase) whilst the cell population is undergoing a growth phase (typically whilst the cell population is in log phase). 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). During step (i), 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. Accordingly, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may continuously detach a proportion (e.g. at least 5%, but no more than 60%) of the cells in the cell population over the culture period. Accordingly, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may continuously detach a proportion (e.g. at least 0.5%, but no more than 7% per hour) of the cells in the cell population over the culture period. In the methods of the invention, 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). In the methods of the invention, 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). As would be clear to a person of skill in the art, “continuously detaching” does not encompass bulk detachment (which is a term that is well known in the art). In the context of metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin), 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. Step (i) of the methods of the invention therefore do not comprise bulk detachment of the cells in the cell population. Suitably, in a culture period, the rate of detachment exceeds the rate of proliferation. Suitably, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) is 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. In other words, 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. Suitably, the detached cells, or a proportion of the detached cells, may be harvested at least once during the cell culture period. Suitably, 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 dead- end or crossflow / tangential fluid flow filtration, a centrifuge, acoustic separation, or a microfluidic based system. Alternatively, detached cells are not harvested, and may remain in the cell culture until after the cell culture period. Before or after step i) of a method of the invention, the cells may be passaged one or more times. Thus, a method may comprise removing cells from a cell culture and placing said cells onto a new support or growth chamber. Suitably, 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. A measure of cell viability may be made after the culture period of step i). Suitably, in a method of the invention, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) does not have a substantial detrimental effect of the viability of the cell population (e.g. over a culture period of at least 24 hours, e.g. at least 48 hours, e.g. at least 72 hours). In this context, 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 a collagenase and / or dispase) and / or a 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. As described in the examples below, cells may also be imaged and analysed using Cytation 1, IoLight, Jiusion USB digital Microscope and ImageJ. Where the method indicates that the cells are more confluent than desired, additional metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be added to the cell culture media to increase the rate of detachment of cells from the support. Account may be taken of the overall concentration of metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin), 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). 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 aggregate, 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. A method of the invention may comprise repeating step i) two or more times. Suitably, the culture period in step i) does not comprise a step of washing the cells (e.g. washing the support comprising the cells). A washing step may be included after step i). Washing may be performed using any suitable method dependent on the choice of cell (and e.g. the choice of support). 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. Suitably, in the cell culture period, 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 metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be used in the present invention. Suitably, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) is compatible with a cell culture media. Suitably, the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) is capable of detaching cells without substantially affecting cell growth or viability. A metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be pure or may contain trace amounts (e.g.15% or less, or 10% or less) of other proteins or enzymes. In one example, the collagenase type I, and as such may contain other enzymes such as caseinase, clostripain and trypsin. Suitably, the collagenase type I may have more than 125U / mg of collagenase; more than 200U / mg of caseinase; less than 4 U / mg of clostripain and less than 0.5U / mg of tryptic activity. Where the metalloprotease 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 GibcoTMCollagenase 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. This is the collagenase type I used in the examples below. A number of 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). Suitably, a single metalloprotease (i.e. collagenase or dispase) or cysteine protease (i.e. papain or ficin) may be used in a cell culture, in a single cell culture period. Alternatively, a combination of two or more proteases that are metalloproteases or cysteine protease may be provided in a cell culture, in a single cell culture period. Merely by way of example one metalloprotease (i.e. collagenase or dispase) and 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. As would be clear to a person of skill in the art, any suitable concentration or amount of metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) may be used, provided that the metalloprotease (such as a collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) continuously detaches a proportion of the cells in the cell population during the culture period. A person of skill in the art can readily determine 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. For example, it is shown that a higher concentration of collagenase was needed for static culture compared to dynamic culture in a flat system, under serum free conditions. Even higher concentrations were needed when microcarriers were used. Further increases in concentration have also been observed when serum was present in the cell culture media (flat-static - steady state continuous detachment achieved with 9 to 20 U / mL of collagenase type I; compared to flat-dynamic - steady state continuous detachment achieved with 5 to 10 U / mL; with bulk detachment requiring 4200 U / mL (20 mg / mL) – data not shown). A suitable concentration may therefore be determined by a person of skill in the art. Suitably, where collagenase is used, it may be provided in a cell culture at or below 500 U / mL. Suitably, it may be provided in a cell culture at or below 215 U / mL. Suitably, the collagenase may be provided in a cell culture at or below 120 U / mL. Suitably, the collagenase may be provided in a cell culture at or below 110 U / mL or at or below 50 U / mL. Suitably, where the collagenase is collagenase type I, it may be provided in a cell culture at or below 215 U / mL. Suitably, the collagenase type I may be provided in a cell culture at or below 120 U / mL. Suitably, the collagenase type I may be provided in the cell culture at or below 110 U / mL. Suitably, the collagenase type I may be provided in the cell culture at or below 80 U / mL. Suitably, the collagenase type I may be provided in the cell culture at or below 50 U / mL. Suitably, the collagenase type I may be provided in the cell culture at or below 20 U / mL. Suitably, the collagenase type I may be provided in the cell culture at or below 10 U / mL. Suitably, the collagenase type I may be provided in the cell culture at or below 5 U / mL. Suitably, the collagenase type I may be provided in the cell culture at or below 3 U / mL. Suitably, the collagenase type I may be provided in the cell culture at or below 2 U / mL. Suitably, 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. In each of these cases the lower end of the range may be 0.005 U / mL e.g.0.008 U / mL. For example, where the collagenase is collagenase type I, it may be provided in a cell culture at a concentration between 0.008 U / mL and 20 U / mL. For example, where the collagenase is collagenase type I, it may be provided in a cell culture at a concentration between 5 U / mL and 20 U / mL, or at a concentration between 9 U / mL and 20 U / mL. For example, where the collagenase is collagenase type I, it may be provided in a cell culture at a concentration between 0.008 U / mL and 10 U / mL. For example, where the collagenase is collagenase type I, it may be provided in a cell culture at a concentration between 5 U / mL and 10 U / mL, or at a concentration between 0.008 U / mL and 5 U / mL. Suitably, where the collagenase is collagenase type VII, it may be provided in a cell culture at or below 500 U / mL. Suitably, the collagenase type VII is provided in a cell culture at or below 300 U / mL. Suitably, the collagenase type VII is provided in a cell culture at or below 200 U / mL. Suitably, the collagenase type VII is provided in a cell culture at or below 150 U / mL. Suitably, the collagenase type VII may be provided in a cell culture at or below 120 U / mL. Suitably, the collagenase type VII may be provided in the cell culture at or below 110 U / mL. In each of these cases the lower end of the range may be 50 U / mL. For example, where the collagenase is collagenase type VII, it may be provided in a cell culture at a concentration between 50 U / mL and 300 U / mL. For example, where the collagenase is collagenase type VII, it may be provided in a cell culture at a concentration between 50 U / mL and 200 U / mL, or at a concentration between 50 U / mL and 150 U / mL. For example, where the collagenase is collagenase type VII, it may be provided in a cell culture at a concentration between 50 U / mL and 120 U / mL. For example, where the collagenase is collagenase type VII, it may be provided in a cell culture at a concentration between 50 U / mL and 110 U / mL. Suitably, where the enzyme is dispase, it may be provided in the cell culture medium at or below 13 U / mL. Suitably, the dispase is present in the cell culture medium at or below 6.5 U / mL. Suitably, the dispase is present in the cell culture medium at or below 3 U / mL. Suitably, the dispase is present in the cell culture medium at or below 2 U / mL or at or below 1 U / mL. Suitably, the dispase is present in the cell culture medium at or below 0.8 U / mL or at or below 0.5 U / mL. In each of these cases the lower end of the range may be 0.0005 U / mL e.g. 0.0008 U / mL. For example, the dispase may be provided in a cell culture at a concentration between 0.0008 U / mL and 3 U / mL. For example, the dispase may be provided in a cell culture at a concentration between 0.0008 U / mL and 2 U / mL, or at a concentration between 0.0008 U / mL and 1 U / mL. For example, the dispase may be provided in a cell culture at a concentration between 0.0008 U / mL and 0.8 U / mL. For example, the dispase may be provided in a cell culture at a concentration between 0.0008 U / mL and 0.5 U / mL. An enzyme unit (U) is a measure of an enzyme’s catalytic activity, and an enzyme unit (U) is the amount of enzyme which catalyses the conversion of one micromole of substrate per minute under the specified conditions. Alternatively, the enzyme activity may be expressed in katals (the enzyme activity which converts one mole of substrate per second under the specified conditions). For collagenase, the enzyme activity unit may be a Collagenase Degrading Unit (CDU or Mandl) 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 specified conditions according to the present invention are those defined in the Examples, for example a static or dynamic serum free culture, on glass microcarriers. An equivalent amount of enzyme units may be determined for different conditions or enzymes by the skilled person by measuring the enzyme activity under different conditions and adjusting the amount of enzyme to achieve the desired level of enzyme activity under the different conditions. For commercially available enzymes, 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. Thus, for any commercially available enzyme, the skilled person can determine the amount of product required to provide the desired enzyme units. By way of example, for collagenase type I, the enzyme units of 110 U / mL is equivalent to 0.5 mg / mL based on the proportion of protease in the product, and the weight of product used in the cell culture. Similarly, the enzyme units of 215 U / mL for collagenase type I as used herein is equivalent to 1 mg / mL. For dispase I as used herein, enzyme units of 6.5 U / mL is equivalent to 0.5 mg / mL and enzyme units of 13 U / mL is equivalent to 1 mg / mL. When expressed as a concentration of enzyme, a metalloprotease (e.g. collagenase and / or dispase) may be provided in the cell culture media at or below 1 mg / mL, or suitably at or below 0.5 mg / mL. Suitably, a protease (e.g. collagenase and / or dispase) may be provided at or below about 1 mg / mL, 0.9 mg / mL, 0.8 mg / mL, 0.7 mg / mL, 0.6 mg / mL, 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, more suitably at or below 0.09 mg / mL, 0.08 mg / mL, 0.07 mg / mL, 0.06 mg / mL, 0.05 mg / mL, 0.04 mg / mL, 0.03 mg / mL, 0.02 mg / mL, 0.01 mg / mL, 0.005 mg / mL, 0.001 mg / mL, or 0.0005 mg / mL. Suitably, the metalloprotease (e.g. collagenase and / or dispase) may be provided at a concentration in the cell culture media of about 0.0001 mg / mL to 1 mg / mL, 0.001 mg / mL to 0.9 mg / mL, 0.01 mg / mL to 0.9 mg / mL, 0.1 mg / mL to 0.9 mg / 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. Suitably, the exogenous cysteine protease (e.g. papain and / or ficin) may be provided at or below about 1 mg / mL, 0.9 mg / mL, 0.8 mg / mL, 0.7 mg / mL, 0.6 mg / mL, 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, more suitably at or below 0.09 mg / mL, 0.08 mg / mL, 0.07 mg / mL, 0.06 mg / mL, 0.05 mg / mL, 0.04 mg / mL, 0.03 mg / mL, 0.02 mg / mL, 0.01 mg / mL, 0.005 mg / mL, 0.001 mg / mL, or 0.0005 mg / mL. Suitably, ficin may be provided at a concentration in the cell culture media of about 0.0001 mg / mL to 1 mg / mL, 0.001 mg / mL to 0.75 mg / mL, 0.001 mg / mL to 0.5 mg / mL, 0.01 mg / mL to 0.25 mg / 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. In another example ficin may be provided at a concentration in the cell culture media of about from 0.005 mg / mL to 0.1 mg / mL, for example of about from 0.01 mg / mL to about 0.05 mg / mL. Suitably, ficin may be provided at a concentration in the cell culture media of about from 0.0001 to about 0.1 BAPA U / mL, 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 / mL. Suitably, ficin may be provided at a concentration of about 0.0033 BAPA U / mL in the cell culture media. Suitably, papain may be provided at a concentration in the cell culture media of about 0.0001 mg / mL to 1mg / mL, 0.001 mg / mL to 0.75 mg / mL, 0.001 mg / mL to 0.5 mg / mL, 0.005 mg / mL to 0.25 mg / 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. In another example papain may be provided at a concentration in the cell culture media of about from 0.0001 mg / mL to 0.07 mg / mL, for example of about from 0.001 mg / mL to about 0.025 mg / mL. Suitably, papain may be provided at a concentration in the cell culture media of from about 0.001 to about 10 TU U / mL, 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 TU U / mL. Suitably, papain may be provided at a concentration of about 0.79 TU U / mL in the cell culture media. 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. For example, a cell culture produced by a method of the present invention may find use in the cultured meat industry. Suitably, a method of the invention may comprise processing steps related to the application of the cells generated in the cell culture. Bulk detachment In another aspect, the present invention provides a method for bulk cell detachment. The method comprises the step of contacting cells with a cysteine protease (such as the cysteine proteases described hereinabove), wherein the cysteine protease bulk detaches the cells. This aspect is based on the inventors surprising that cysteine proteases can be used instead of traditional serine proteases to bulk detach cells. The cysteine proteases exemplified in the present application are plant proteases, i.e. proteases that naturally exist in plants. It is believed that the use of such plant proteases in the context of animal cell culture is novel and unexpected. It will be appreciated that for such bulk detachment, the concentration of the cysteine protease is higher in the cell culture medium as compared to the concentration used for continuous cell detachment. Suitably, in the context of bulk cell detachment, the concentration of the cysteine proteases (such as papain and / or ficin) may be above 1 mg / mL. For example, the concentration may be above 1.5 mg / mL, above 2 mg / mL, above 3 mg / mL, above 4 mg / mL, above 5 mg / mL, above 10 mg / mL, above 15 mg / mL, above 20 mg / mL, above 30 mg / mL, above 35 mg / mL. Suitably, in the context of bulk cell detachment, the concentration of the cysteine proteases (such as papain and / or ficin) may be from about 1 mg / mL to about 100 mg / mL, for example from about 1.5 mg / mL to about 50 mg / mL, or from about 1.5 mg / mL to about 40 mg / mL. Suitably, in the context of bulk cell detachment, the concentration of the ficin may be above 0.1 BAPA U / mL, for example above 0.3 BAPA U / mL, or above 0.3 BAPA U / mL. Suitably, the concentration of the ficin may be from about 0.1 BAPA U / mL to about 10 BAPA U / mL, for example from about 0.3 BAPA U / mL to about 0.5 BAPA U / mL, or from about 0.39 BAPA U / mL to about 6.3 BAPA U / mL. Suitably, in the context of bulk cell detachment, the concentration of the papain may be above 100 TU U / mL, for example above 200 TU U / mL, Suitably, the concentration of the papain may be from about 100 TU U / mL to about 10000 TU U / mL, for example from about 200 TU U / mL to about 5000 TU U / mL, or from about 200 TU U / mL to about 4000 TU U / mL. Suitably, the concentration of the papain may be from about 236 TU U / mL to about 3772 TU U / mL. Suitably, the cells may be contacted with the cysteine protease for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes or more. For example, the cells may be contacted with the cysteine protease for at least 40 minutes, for at least 50 minutes, for at least 60 minutes, for at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes or more. For example at least 120 minutes or 150 minutes. Suitably, the cells may be contacted with the cysteine protease from for about 10 minutes to 200 minutes, for example from about 20 minutes to about 175 minutes, or from about 30 to about 150 minutes. It will be clear to a person of skill in the art, that a higher concentration of the cysteine protease may enable a shorter time of contacting the cells with the enzymes. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties, or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. EXAMPLES Example 1 Data have been generated using C2C12 as cell model grown in serum-free media (DMEM F12 with 1% Insulin Transferrin Factor and 1mM of L-Ascorbic Acid). 1.1. Collagenase can successfully detach C2C12 cells in bulk without altering their adhesion, proliferation, and differentiation potential. Collagenase has previously been used for the bulk detachment of some types of adherent cells in alternative to more common methods such as trypsin and TrypLE. In order to achieve bulk cell detachment in short periods of time a high concentration of the collagenase was added and incubated at 37°C for 30 min (pre-warmed collagenase solution before addition). Collagenase type I concentration used: 1180U (or 472 U / mL; which is equivalent to 2.2 mg / mL). Collagenase supplementation showed comparable results to TrypLE in terms of detachment efficiency (Figure 1A) and viability (Figure 1B) of the collected cells after detachment. Moreover, similarly to the control cells, the collagenase-detached cells can reattach, proliferate, and differentiate (Figure 1C-E). 1.2. Collagenase is capable of detaching cells in a dose-dependent manner. It was demonstrated that increasing amounts of collagenase lead to the detachment of an increasing number of C2C12 cells (Table 1). Table 1: Quantification showing increasing number of detached cells using Collagenase type I (units / cell) in a dose-dependent manner. Collagenase Collagenase Collagenase Detached Number of [Units / cell] [Units / mL] [mg / mL] Cells 3.92x10-52.625 0.012 445517 5.22x10-53.500 0.016 572663 6.53x10-54.375 0.020 594022 This suggested that collagenase could surprisingly be used to control the rate of cell detachment. Moreover, the detached cells were capable of reattaching and were not differentiated during the process (Figure 2). 1.3. Low concentrations of collagenase allow for continuous detachment of C2C12 cells. It was demonstrated that dosing the collagenase at lower concentrations allowed for continuous cell detachment without impacting cell characteristics. This was investigated in 4 different systems (see A to D below). A. Continuous cell detachment in a flat system under static conditions. It was demonstrated that supplementing with 1.75 U / mL (equivalent to 0.0081 mg / mL of the Collagenase type I used herein) of collagenase type I allowed C2C21 cells to continuously grow and detach for 21 days in static culture in serum-free media (Figure 4). Detached cells were collected every 24 hours and counted at day 1, 7, 9, 14 and 21. Over the time in culture, the confluence on the growing surface remained similar (Figure 4A) without growing to over- confluency levels. Each surface with growing cells was moved to a different (new) well every 48h and fresh media and collagenase was added. Cells detached from the surface at similar levels over the time (Figure 4B). B. Continuous detachment in a flat system in dynamic conditions. It was demonstrated that supplementing with 0.009 U / mL (equivalent to 0.00004 mg / mL (43.27 ng / mL) of the Collagenase type I used herein) of collagenase type I allowed C2C12 cells to continuously grow and detach for 7 days in dynamic culture in serum-free media (Figure 5). Detached cells were collected at the end of the experiment whilst growing cells were imaged every day (Figure 5A). At the end of the experiment, the number of harvested attached cells was similar to the number of seeded cells (Figure 5B). In addition, both attached and detached cells displayed the initial undifferentiated phenotype at the end of the 7 days continuous culture under flow (Figure 5C&D). C. Continuous detachment in microcarrier system in static conditions. It was demonstrated that supplementing with 4.5 U / mL of collagenase type I allowed C2C12 cells to continuously grow and detach for 27 days in static culture in serum-free media. The confluence of the growing cells on the microcarriers was maintained during the time in culture and cell detachment was observed after supplementation of collagenase started at day 1 (Figure 6A). The detached cells were collected every 24h and the average number of cells that detached were consistent between the first and the second week (Figure 6D), with around a third of the initially seeded cells detached every 24h. The number of cells that were harvested from (previously growing on) the microcarriers did increase from day 1, however the cells did not overgrow on the microcarrier surface leading to aggregations (Figure 6E). Moreover, both (i) the detached (yield) cells that detached during the process and (ii) the attached cells recovered from (growing on) the microcarriers at day 27, were capable of reattaching to the tissue culture plastic after collection and displayed the initial undifferentiated phenotype (Figure 7A). In addition, the detached (yield) cells were capable of re-attaching to fresh microcarriers in presence of collagenase and undergo the same process again (Figure 7B). D. Continuous detachment in microcarriers system in dynamic condition. It was demonstrated that supplementing with 4.5 U / mL of collagenase type I allowed C2C12 cells to continuously grow and detach for 27 days in dynamic culture in serum-free media. Cell detachment was observed throughout the time in culture with collagenase supplementation (Figure 8). Detached cells were collected every 24h (Figure 8A) and the average number of cells that detached has been consistent between the four weeks (Figure 8B), with around a third of the initially seeded cells detaching every 24h. The number of cells that were harvested from (previously growing on) the microcarriers did increase from day 1, however these cells did not overgrow on the microcarrier surface (Figure 8D). As observed in the static system, both (i) detached (yield) cells that detached during the process and (ii) attached cell recovered from (growing on) the microcarriers at day 27, were capable of reattaching to the tissue culture plastic after collection and displayed the initial undifferentiated phenotype (Figure 9A). Finally, the detached (yield) cells were capable of remaining functional after being cryopreserved for a few days (Figure 9B). Dispase I Different concentrations of dispase I were tested on C2C12 cells grown in serum-free culture media. Bulk detachment was achieved supplementing the media with 0.8 U / mL (equivalent to 0.062 mg / mL) of dispase I used herein. With serial dilutions starting from 0.8 U / mL (equivalent to 0.062 mg / mL) of dispase I, the inventors found that 0.0008 U / mL (equivalent to 0.00006 mg / mL (60 ng / mL)) allowed them to continuously detach C2C12 over a period of 5 days. This preliminary experiment suggests that dispase I could be used as an alternative to Collagenase type I (see Figure 11). Collagenase VII (pure collagenase) The inventors also tested pure collagenase (collagenase VII) to understand if it could also be used to achieve continuous cell detachment. A wide range of concentrations were tested, and continuous cell detachment was achieved using higher enzyme units compared to the crude reagent (collagenase type I). An optimal concentration for Collagenase VII was determined to be in the range between 106.35 U / mL and 53.18 U / mL [see figure 12]. Thus, pure collagenase is also able to continuously detach cells. Example 2 This example provides data supporting that cysteine proteases Ficin and Performase® can be used similarly to collagenase and dispase for continuous detachment of adherent cells. Data have been generated using C2C12 as cell model grown in serum-free media (DMEM F12 with 1% Insulin Transferrin Factor and 1mM of L-Ascorbic Acid). The data provided below shows that Ficin and Performase® can: i) detach cells in a dose-dependent manner; ii) detach cells at steady-state without altering cell phenotype; and iii) detach cells in bulk without compromising cell viability. The two plant-based cysteine proteases that were tested are Ficin (product FSM200) and Performase® (product PSM100) purchased from Enzybel International S.A. Ficin and Papain (Performase® is derived from Papain) are cysteine proteases (they have a sulfhydryl group at their active site). Ficin Ficin (historically called Ficain) was purified from the latex of fig tree Ficus glabrata or Ficus anthelmintica and is part of Cysteine endopeptidase family. This Ficin is a food-grade natural protease. Optimum working conditions are pH 5-9 and Temp 40-75°C. The CoA (Certificate of Analysis) from Enzybel stated the active units of this batch as 213 BAPA / g (*). Such enzyme is already used for food production and pharmaceuticals applications. Performase® Performase® is an enzyme preparation derived from papaya (peptidase-papain); it is food grade and containing endopeptidase. The CoA from Enzybel stated active units of this batch as 103 TU / mg (**). Performase® is already used for food & beverages, and pharmaceuticals applications. Origin: Garica papaya L. of the BAPA Method: For the analysis of enzymatic (proteolytic) activity of Ficin, a synthetic substrate is used N- α-benzoyl-DL-arginine-p-nitroanilide hydrochloride (DL-BAP(N)A), which is cleaved by the enzyme. The substance "p-nitroanilin" (cleaved fragment) gives an indication on the hydrolysis rate during the reaction. This substance is measured photometrically. One unit of Ficin activity corresponds with the quantity of enzyme that hydrolyses the equivalent of 1 μmol of the substrate per minute under the conditions of the assay. **Principle of the TU Method: Enzyme solution added to a buffered solution of casein which is digested at 40°C and pH 6.0 for 60 minutes. The reaction is stopped, and the excess of casein is precipitated with trichloracetic acid. The digest is filtered, and the optical density of the clear filtrate is read at 280 nm. The activity of the enzyme is then calculated from the observed optical density and the slope of a Standard Tyrosine curve. TU unit (TU / mg) is defined as the unit which, while acting on a casein substrate under specific conditions, releases 1μg of Tyrosine per minute. 2.1 Dose-dependent cell detachment Similarly, to collagenase and dispase, both Ficin and Performase® are capable of detaching cells in a dose-dependent manner. Hence, increasing concentrations of enzyme detach an increased number of cells from the surface. Figure 13 shows images of cells growing on surfaces after 6 days of incubation with different enzyme concentrations. The concentrations of Ficin (Figure 13A) used were 0.06 µg / mL, 0.6 µ / mL, and 60 µg / mL. The concentrations of Performase® (Figure 13B) used were 0.07 µg / mL, 0.7 µg / mL, and 70 µg / mL. As the enzyme's concentrations increased, the number of detached cells increased (numerical data not shown, but observable in Figure 13) leaving less cells growing on the original surface. Hence, there is a dose-dependency between the enzyme concentration and the number of detached cells. This was confirmed for both enzymes (Figure 13). 2.2 Continuous cell detachment Similarly, to collagenase and dispase, both Ficin and Performase® are capable of detaching cells at a steady-state. Gathered data shows continuous growth and detachment of C2C12 cells over 10 days in a static system in serum-free media (Figure 14) Images in Figure 14 shows (A) Ficin at 15.36 µg / mL (0.0033 BAPA U / mL) and (B) Performase® at 7.68 µg / mL (0.79 TU U / mL) were capable to maintain over 10 days a similar confluency on the proliferating surface (attached) whilst cells were continuously detaching from it (detached). Moreover, immunostaining shows that the process did not interfere with the phenotype of both attached and detached cells. Indeed, Pax7 positivity was found above 90% in all conditions (data not shown). 2.3 Bulk cell detachment Similarly to collagenase and dispase, both Ficin and Performase® can detach cells in bulk once added to the cell culture media. In this experiment C2C12 cells were seeded at a density of 50,000 / cm2in a 24 well plate with 1mL of serum free media (SFM). Cells were allowed to adhere to the surface overnight. Each enzyme was then prepared in SFM and 1mL added to each well at concentrations ranging from 29.6 mg / mL (6.3 BAPA U / mL) to 1.85 mg / mL (0.39 BAPA U / mL) for ficin and between 36.625 mg / mL (3772 TU U / mL) to 2.29 mg / mL (236 TU U / mL) for Performase®. Both enzymes (Ficin at 29.6 mg / mL (i.e.6.3 BAPA U / mL) and Performase®, 36.625 mg / mL (i.e.3772 TU U / mL)) at the tested concentrations could efficiently detach cells from the surface within 30 minutes after their addition (Figure 15). Complete detachment occurred after 150 minutes (Figure 15) for both enzymes. Cell viability Moreover, the addition of Ficin and Performase® did not compromise cell viability during the total 4h of incubation with the enzymes. The detached cells were collected, centrifuged, and resuspended in serum-free media (SFM) for cell count & viability. Viability of such detached cells was found to be 90.5% and 98.5% when detached with Performase® and ficin, respectively (Figure 16). Conclusion Ficin and Performase® are capable of continually detaching adherent cells at a steady-state without altering cell viability and phenotype when added at low concentrations to the culture media.

Claims

CLAIMS 1. A method of cell culture of an adherent cell population, comprising: (i) culturing the cell population in a cell culture medium comprising a metalloprotease and / or cysteine protease for a culture period which comprises all or part of the log phase of growth of the cell population, wherein the metalloprotease and / or cysteine protease continuously detaches a proportion of the cells in the cell population.

2. A method of cell culture of an adherent cell population, comprising: (i) culturing the cell population in a cell culture medium comprising a metalloprotease and / or cysteine protease for a culture period which is at least 24 hours, wherein the metalloprotease and / or cysteine protease continuously detaches a proportion of the cells in the cell population.

3. The method of claim 1 or 2, wherein the metalloprotease is a collagenase and / or a dispase.

4. The method of claim 1 or 2, wherein the cysteine protease is of the CA clan, optionally selected from the group consisting of ficin, papain, bromelain, cathepsin K and calpain, further optionally selected from the group consisting of ficin and papain.

5. The method of claim 4, wherein the collagenase is collagenase type I or collagenase type VII.

6. The method of claim 3, wherein the dispase is dispase I.

7. The method of any preceding claim, wherein step (i) is performed for at least 3 days.

8. The method of any preceding claim, wherein the cell population is cultured on a support in the cell culture medium, wherein the collagenase and / or dispase continuously detaches a proportion of the cells in the cell population from the support.

9. The method of claim 8, wherein the support comprises a material selected from the group consisting of: plastic, polymer, glass, and metal.

10. The method of claim 8 or 9, wherein 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.

11. The method of any one of claims 1 to 7, wherein the cell population is a cell aggregate, wherein the metalloprotease and / or cysteine protease continuously detaches a proportion of the cells from the cell aggregate.

12. The method of any preceding claim, wherein the cell population comprises cells selected from the group consisting of: skin, muscle, cervical, breast, and prostate cells.

13. The method of any preceding claim, wherein the method comprises culturing the cell population in a cell culture medium lacking the metalloprotease and / or cysteine protease before step (i).

14. The method of any preceding claim, further comprising adjusting the metalloprotease and / or cysteine protease concentration or frequency to adjust the rate of cell detachment, wherein increasing the concentration or frequency increases the rate of continuous cell detachment and decreasing the concentration or frequency decreases the rate of continuous cell detachment.

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