Cell culture of adherent cells

Abstract

The present invention relates to a method for the continuous culture of adherent cells.

Description

Technical Field

[0001] The present invention relates to a method for the continuous culture of adherent cells. Background Art

[0002] Regenerative medicine, and cell therapy in particular, has been expanding its scope in targeted cell types and applications, increasing its potential to cure a wide range of diseases. However, the high cell counts required for some therapies (which can be as high as 1 billion) present significant challenges in cell manufacturing.

[0003] Furthermore, because the final product is often represented by the cells themselves, numerous requirements must be met under the current regulatory framework for cell therapy products.

[0004] Therefore, in order to sustain this huge demand for cells and meet the required conditions, attempts have been made to implement and adapt industrial processes previously developed for large-scale mammalian cell culture production of biologics.

[0005] While significant increases in cell yields have been achieved, several other key production steps are still being optimized. For example, improved cell harvesting methods are needed to more easily, efficiently, and less invasively recover expanded cells from their culture matrix.

[0006] When adherent cells are grown in culture, the first stage of growth is called the lag phase, in which cells are seeded into a growth chamber (e.g., onto a support or in a culture medium to grow as cell aggregates), where they need time to adapt to their new environment and prepare for rapid growth. The subsequent logarithmic phase is the period during which cells grow exponentially and consume the nutrients in the cell culture medium. When the growth medium is exhausted, or when cells reach confluence, the cells enter the stationary phase. At this stage, cell proliferation is greatly reduced or may stop completely.

[0007] In traditional cell culture methods, cells are harvested in large quantities after they enter the stationary phase. The discarded cell culture medium 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 the cells and growing them in the lag phase is then repeated until logarithmic growth can be achieved. This traditional cell culture method is a batch process.

[0008] The object of the present invention is to improve the recovery of cells from cell culture and thereby ameliorate some of the problems associated with the prior art. Summary of the Invention

[0009] In one aspect of the present invention, a method for culturing an adherent cell population is provided, the method comprising:

[0010] (i) culturing the cell population in a cell culture medium comprising a metalloprotease and / or a cysteine protease for a culture period that includes all or a portion of a logarithmic growth phase of the cell population, wherein the metalloprotease and / or the cysteine protease continuously detaches a portion of the cells in the cell population. In this context, the cell culture medium can be said to be supplemented with the metalloprotease and / or the cysteine protease.

[0011] In other words, in one aspect of the present invention, a method for culturing an adherent cell population is provided, the method comprising:

[0012] (i) culturing the cell population in a cell culture medium comprising an exogenous metalloprotease and / or an exogenous cysteine protease for a culture period that includes all or a portion of a logarithmic growth phase of the cell population, wherein the exogenous metalloprotease and / or the exogenous cysteine protease continuously detaches a portion of the cells in the cell population.

[0013] Suitably, the incubation period may be at least 24 hours.

[0014] 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 family.

[0015] Suitably, the cysteine protease of the CA family may be selected from the group consisting of: ficin, papain, bromelain, cathepsin K and calpain. More suitably, the cysteine protease of the CA family may be ficin and / or papain.

[0016] In another aspect of the present invention, a cell culture method for an adherent cell population is provided, the method comprising:

[0017] (i) culturing the cell population in a cell culture medium comprising a metalloprotease and / or a cysteine protease for a culture period of at least 24 hours, wherein the metalloprotease and / or the cysteine protease continuously detaches a portion 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 the cysteine protease.

[0018] In other words, in another aspect of the present invention, a cell culture method for an adherent cell population is provided, the method comprising:

[0019] (i) culturing the cell population in a cell culture medium comprising exogenous metalloproteases and / or cysteine proteases for a culture period of at least 24 hours, wherein the metalloproteases and / or cysteine proteases continuously detach a portion of the cells in the cell population.

[0020] Suitably, in the methods of the invention, the culture period comprises all or part of the logarithmic growth phase of the cell population.

[0021] Suitably, in the methods of the present invention, the cell culture medium may comprise a metalloprotease.

[0022] Suitably, in the methods of the present invention, the cell culture medium may comprise collagenase.

[0023] Suitably, in the method of the present invention, the collagenase is a collagenase selected from the group consisting of type I collagenase, type II collagenase, type III collagenase, type IV collagenase, type V collagenase, type VI collagenase and type VII collagenase. Notably, type I to type VII collagenases are enzyme compositions comprising collagenases of increasing purity, wherein type VII collagenase is a pure collagenase.

[0024] Suitably, in the method of the present invention, the collagenase is type I collagenase or type VII collagenase. These are the collagenases used herein to illustrate the present invention, but the present invention is not limited thereto.

[0025] Suitably, in the methods of the invention, the cell culture medium comprises dispase.

[0026] Suitably, in the method of the present invention, the dispase is a type I dispase or a type II dispase.

[0027] Suitably, in the methods of the invention, the cell culture medium may comprise a cysteine protease. Suitably, the cysteine protease may be of the CA family.

[0028] Suitably, the cysteine protease of the CA family may be selected from the group consisting of ficin, papain, bromelain, cathepsin K and calpain.

[0029] More suitably, in the method of the present invention, the cysteine protease of the CA family may be ficin and / or papain.

[0030] Suitably, in the methods of the invention, the metalloprotease and / or cysteine protease is provided in the cell culture medium at a concentration that allows a ratio of the detachment rate of the cells (e.g. detachment from a support or from other adherent cells, such as from cell aggregates) to the growth rate of the cells in the range of 0.3:1 to 1:3.

[0031] Suitably, in the methods of the invention, the metalloprotease and / or cysteine protease is provided in the cell culture medium at a concentration that maintains the cell culture at about 50% to 100% confluence.

[0032] Suitably, in the methods of the invention, the detached cells or a portion of the detached cells may be harvested at least once during the cell culture period. Suitably, in the methods of the invention, the detached cells or a portion of the detached cells may be harvested more than once during the cell culture period.

[0033] Suitably, in the methods of the invention, the incubation period does not comprise a step of washing the cells (eg it does not comprise a step of washing the support comprising the cells, or a step of washing cell aggregates comprising the cells).

[0034] Suitably, in the methods of the present invention, the cell culture medium is supplemented with a metalloprotease and / or a cysteine protease one or more times during the cell culture period.

[0035] Suitably, in the method of the invention, a cell population is cultured on a support in a cell culture medium, wherein the metalloprotease and / or cysteine protease continuously detaches a portion of the cells in the cell population from the support.

[0036] Suitably, in the methods of the present invention, the support comprises a material selected from the group consisting of: plastic, polymer, glass and metal.

[0037] Suitably, in the method of the invention, the support is selected from the group consisting of beads, microcarriers, microfluidic chips, silicon chips, microscope slides, microplate wells, matrices, resins, biochips, multiwell plates, gels, films and membranes.

[0038] Suitably, in the method of the invention, the cell population is a cell aggregate, wherein the metalloprotease and / or cysteine protease continuously detaches a portion of the cells from the cell aggregate.

[0039] Suitably, in the methods of the present invention, the cell population comprises cells selected from the group consisting of skin cells, muscle cells, cervical cells, breast cells and prostate cells.

[0040] Suitably, the method of the invention comprises culturing the cell population in a cell culture medium lacking a metalloprotease and / or a cysteine protease prior to step (i).

[0041] Suitably, the methods of the invention comprise modulating the concentration and / or frequency of administration of the metalloprotease and / or cysteine protease to modulate the rate of cell detachment, wherein increasing the concentration and / or frequency of administration increases the rate of continuous cell detachment, and decreasing the concentration and / or frequency of administration decreases the rate of continuous cell detachment.

[0042] In a third aspect of the present invention, a cell culture container is provided, comprising a cell culture medium, an adherent cell population in a logarithmic growth phase, and a metalloproteinase and / or a cysteine protease.

[0043] Suitably, the cell culture vessel comprises a metalloprotease.

[0044] Suitably, the metalloprotease is collagenase and / or dispase.

[0045] Suitably, the metalloprotease is collagenase.

[0046] Suitably, the collagenase is a collagenase selected from the group consisting of type I collagenase, type II collagenase, type III collagenase, type IV collagenase, type V collagenase, type VI collagenase and type VII collagenase.

[0047] Suitably, the collagenase is type I collagenase or type VII collagenase.

[0048] Suitably, the metalloprotease is dispase.

[0049] Suitably, the dispase is a Type I dispase or a Type II dispase.

[0050] Suitably, the cell culture vessel comprises a cysteine protease.

[0051] Suitably, the cysteine protease may be of the CA family.

[0052] Suitably, the cysteine protease of the CA family may be selected from the group consisting of ficin, papain, bromelain, cathepsin K and calpain.

[0053] More suitably, the cysteine protease of the CA family may be ficin and / or papain. Suitably, the cell population is on a support in a cell culture medium.

[0054] Suitably, the support comprises a material selected from the group consisting of plastic, polymer, glass and metal.

[0055] Suitably, the support is selected from the group consisting of beads, microcarriers, microfluidic chips, silicon chips, microscope slides, microplate wells, matrices, resins, biochips, multiwell plates, gels, films and membranes.

[0056] Suitably, the cell population is a cell aggregate.

[0057] Suitably, the cell population comprises cells selected from the group consisting of skin cells, muscle cells, cervical cells, breast cells and prostate cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Embodiments of the present invention are further described below with reference to the accompanying drawings, in which:

[0059] Figure 1This figure shows that collagenase can successfully detach C2C12 cells in bulk, similar to TrypLE (illustrated using collagenase type I). The figure shows that the number (A) and viability (B) of detached cells using TrypLE and collagenase are similar, without altering cell adhesion (C), proliferation (D), and differentiation (E) potential (scale bar 200 μm).

[0060] Figure 2 It is shown that collagenase can detach cells in a dose-dependent manner without changing the cell phenotype (illustrated using type I collagenase). Images show attached (A) and detached (B) cells in the presence of different concentrations of collagenase. The amount of collagenase (U / mL) is shown in the figure. The equivalent amounts of type I collagenase (in mg / mL) 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) The graph shows that cell detachment increases with increasing collagenase concentration. (D) Representative images show that at the end of the experiment, only undifferentiated cell markers (Pax7) are expressed in attached cells, but not differentiated cell markers (MHC) (scale bar 200 μm).

[0061] Figure 3 Schematic diagram showing collagenase supplementation as a strategy for continuous detachment of adherent cells in two systems: on planar and microcarrier surfaces and under two conditions (static and dynamic). Type I collagenase is used for illustration.

[0062] Figure 4 Continuous detachment is shown in a planar-static system. Cell detachment using 1.75 U / mL (equivalent to 0.008 mg / mL) collagenase (type I collagenase) shows similar numbers of attached (A) and detached (B) cells over 3 weeks. Quantification shows similar numbers of attached (C) and detached (D) cells over time, indicating steady-state cell proliferation and cell yield (scale bar 200 μm).

[0063] Figure 5 Continuous detachment in a planar dynamic system is shown. This is illustrated using type I collagenase. (A) Images show similar numbers of attached cells over the course of a week in the planar dynamic system. (B) Quantification shows that the number of cells harvested on day 7 is similar to that of cells seeded on day 0. (C & D) Representative images show expression of the undifferentiated cell marker Pax7 in detached (C) and attached (D) cells (scale bar 200 μm).

[0064] Figure 6Shows continuous detachment of cells from the microcarrier surface when the culture medium is supplemented with collagenase (illustrated using type I collagenase, A). Stained nuclei show microcarrier aggregation caused by cell overgrowth in the control (B), while the addition of collagenase inhibits microcarrier bridging and cell overgrowth (C). Quantification shows (D) the average number of cells detached per week (over 27 days) using collagenase in a static system and (E) the total number of cells harvested from the microcarriers at the end of the experiment (scale bars 200 μm and 1000 μm).

[0065] Figure 7 Demonstrating the behavior of attached cells. Cells detached from microcarriers using collagenase (A) can reattach to a flat tissue culture-treated surface in serum-containing medium, and cells recovered from microcarriers at the end of the experiment (B). (C) Cells detached using collagenase can also attach to fresh microcarriers and maintain this process in serum-free medium containing collagenase. Illustrated using type I collagenase (scale bar 200 μm).

[0066] Figure 8 Demonstrating continuous detachment in a dynamic microcarrier system. Quantification of the number of cells detached daily (A) and weekly (B) over 27 days using collagenase in a dynamic system. (C) Representative images of nuclear-stained cells on microcarriers and (D) final quantification showing the number of cells seeded and harvested at the end of the experiment. Example using type I collagenase (scale bars 200 μm and 1000 μm).

[0067] Figure 9 Figure 2. Attached cell behavior. Cells detached from microcarriers using collagenase (detachment, A) can reattach to a flat tissue culture treated surface in serum-containing medium, and cells recovered from microcarriers at the end of the experiment (attachment, B). (C) Cells detached from collagenase can be cryopreserved and are able to reattach and grow on tissue culture treated plastic after thawing (thawed cryopreservation). Illustrated using type I collagenase (scale bar 200 μm).

[0068] Figure 10 Demonstrating that collagenase can successfully detach fish skin fibroblasts in bulk. Cells were detached after 20 hours of incubation with collagenase (A), and after collection (B), they were able to reattach (5 hours after seeding, C) and proliferate (48 hours after seeding, D). This example uses type I collagenase at a concentration of 1505 U / mL (equivalent to 7 mg / mL, scale bar 500 μm).

[0069] Figure 11Dispase was shown to successfully and continuously detach C2C12 cells. Confluence of growing cells was maintained over a 3-day period (attachment, A), while cells were detached at a comparable rate over time (detachment, B). Further experiments also showed the same effect over 21 days (data not shown). This was illustrated using Type I Dispase at a concentration of 0.0008 U / mL (equivalent to 0.00006 mg / mL or 60 ng / mL, scale bar 200 μm).

[0070] Figure 12 Pure collagenase was shown to successfully and continuously detach C2C12 cells. Confluence (attachment) of growing cells was maintained over a 3-day period, while cells were detached (detached) at a comparable rate over time. This was illustrated using type VII collagenase. (A) Type VII collagenase was used at a concentration of 106.35 units / mL (equivalent to 0.07 mg / mL); (B) Type VII collagenase was used at a concentration of 53.18 units / mL (equivalent to 0.035 mg / mL, scale bar 200 μm).

[0071] Figure 13 Showing ficin and Cells detached in a dose-dependent manner. Images show the detachment of cells with different concentrations of ficin (A) and (B) C2C12 cells grown on the surface after 6 days of incubation (scale bar 250 μm).

[0072] Figure 14 Shown with ficin (A) or (B) Continuous cell detachment. Images taken on days 0 and 10 show the initial confluence (after cell seeding) and confluence after continuous enzyme exposure, respectively. The day 10 image shows the confluence on the growth surface (attachment) and cells detached during the last 3 days of the experiment (detachment, scale bar 250 μm).

[0073] Figure 15 Shown with ficin or Once added to the culture medium, C2C12 cells can be treated with ficin (A) and (B) Bulk detachment. Cells detached after 30 min, and complete detachment occurred after 150 min (scale bar 250 μm).

[0074] Figure 16 Shown in ficin or Cell viability after exposure. The detached C2C12 cells maintained high cell viability after exposure to high enzyme concentration for 4 h. DETAILED DESCRIPTION

[0075] definition

[0076] 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 the use of the term is unclear to persons of ordinary skill given the context in which it is used, "about" will mean plus or minus 10% of the specified value.

[0077] As used herein, the terms "comprise," "comprises," and "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components of aspects or embodiments of the present invention, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or combinations thereof.

[0078] The phrase "consisting essentially of means that the scope of an aspect or embodiment should be interpreted to encompass the specified materials or steps recited in the aspect or embodiment, including any materials or steps that do not materially affect the invention as defined by the aspect or embodiment. Therefore, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising."

[0079] As referred to herein, a "support" is any surface suitable for supporting biological material, such as a cell, cell population, cell culture, tissue, or fluid or other biological material or composition, such as described herein. A support can be referred to as a surface that can be suitable for accommodating a process or reaction, such as the growth and development of a cell or organism, cell population, cell culture, or tissue.

[0080] A "gel" is a semisolid, colloidal substance that does not flow when in the solid state. A gel contains a 3D cross-linked network that provides the gel with its semisolid structure. A gel can be a hydrogel, which contains a network of insoluble but hydrophilic polymer chains. A gel can be defined based on its viscoelastic or rheological properties.

[0081] A buffer is a solution that resists significant changes in pH after the addition of a small amount of acid or base. A buffer is a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid.

[0082] As used herein, "cell viability" refers to the ability of a cell to remain metabolically active in order to grow and function.

[0083] The "culture period" of cells is the period of time that cells are maintained in cell culture under conditions suitable for growth.

[0084] As used herein, "confluence" in the context of a support refers to the percentage of the support covered by adherent cells. 50% confluence means that half of the support is covered by adherent cells. 100% confluence, or complete confluence, means that the entire support is covered by adherent cells. Overconfluence means that there is no space available on the support for new cells.

[0085] In the context of cell aggregates, cell density or aggregate size is more relevant. Methods for determining cell density are described elsewhere herein.

[0086] As used herein, the term "cell culture medium" refers to a nutrient solution used to culture living cells for the purpose of cell proliferation.

[0087] The term "contacting" refers to bringing two or more items into contact with each other. The items may be two or more of a cell, a support, and / or a cell culture medium, as suitably defined herein. Contacting may include placing or bringing two or more of the above items into close physical relationship and / or in contact with each other. Contacting may sometimes be referred to as "exposing," e.g., exposing a cell to a cysteine protease should be understood as bringing the cell into contact with the cysteine protease.

[0088] As used herein, the term "adherent cells" refers to a homogeneous or heterogeneous population of cells that are anchorage-dependent, ie, require attachment to a support or other adherent cells (eg, in the form of cell aggregates) in order to grow in vitro.

[0089] The term "cell aggregate" may also be referred to herein as a multicellular aggregate. An aggregate refers to, for example, a ball, cluster, layer, or the like of cells. It refers to a plurality of adjacent or interconnected cells. A cell aggregate can be formed, for example, by at least 10 adjacent cells (wherein each cell is in direct contact (in other words, in contact) with at least one other cell in the aggregate). For example, an aggregate can contain at least 10, at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 or at least 10 9 In a preferred embodiment, the adjacent cells are interconnected.

[0090] As used herein, "adjacent " refers to cells connected to each other in the mode of forming cell aggregates.When placed in a solution (such as a cell culture medium), adjacent cells maintain aggregate form.Adjacent cells can directly contact, for example, wherein they adhere to or contact each other in the mode of forming cell aggregates. Alternatively, adjacent cells can be indirectly connected in the mode of forming cell aggregates, such as by means of the presence of matrix, support or support (such as extracellular matrix), wherein matrix, support or support connect adjacent cells into aggregate.

[0091] As mentioned above, matrix, substrate or scaffold can connect adjacent cells to form aggregate. The terms "matrix", "substrate" and "scaffold" used interchangeably herein are generally referred to as "structure" within the aggregate. Structure can also promote or maintain aggregate formation. Structure can be natural origin or synthetic. In an example, structure can be synthetic or natural polymer. Preferably, structure is biodegradable. Structure can be a polymer that for example comprises polylactic acid (for example poly (lactic acid-co-caprolactone) (PLACL)), collagen or nylon.

[0092] In another example, cells are adjoined by an extracellular matrix (ECM) to form multicellular aggregates. Another example of a suitable structure is a 3D cell culture Polystyrene scaffold. Other structures can contain collagen, gelatin, alginate, cellulose, glass, or matrigel. The structure can also be a nylon mesh.

[0093] Alternatively, the aggregates may be unstructured. Suitable methods for culturing cells with or without structure are well known in the art.

[0094] In a preferred embodiment, adjacent cells are interconnected. As used herein, "interconnected" refers to cells that are in direct contact with each other and, for example, physically connected by intercellular connections (e.g., by one or more cell connections (also referred to as one or more intercellular bridges)). Cell connection is composed of a multiprotein complex that provides contact between adjacent cells or between a cell and an extracellular matrix. Cell connection is particularly abundant in epithelial tissue. Cell connection enables communication between adjacent cells.

[0095] The cell aggregate can be any group of adjacent cells, for example, it can be in the form of a tissue or organ (for example, an animal or plant tissue or organ, or a synthetic / artificial tissue or organ, i.e., a tissue-engineered tissue or organ). Examples of suitable animal tissues or organs include skin, cornea, muscle, liver, and heart tissue or organ. Such tissues or organs can be obtained directly from living animals. Methods for separating suitable multicellular aggregates from animals are well known in the art. Examples of suitable plant tissues or organs (obtained from living plants) include cells or tissues derived from endoderm, mesoderm, and ectoderm germ layers, mesophyll tissue, xylem tissue, and phloem tissue, leaves, stems, roots, and reproductive organs. Methods for separating suitable multicellular aggregates from plants are well known in the art.

[0096] Examples of suitable synthetic tissues or organs include any cell tissue or organ that has been generated or propagated in vitro or ex vivo. Non-limiting examples include cell spheres, spheroids, organoids, or microtissues.

[0097] In one example, cell aggregates can be attached to the surface of a vessel (eg, a culture vessel) in which they are seeded and / or grown in vitro.

[0098] In a particular example, a cell aggregate comprises a plurality of contiguous (eg, interconnected) cells, wherein the cells form a tissue, a cell layer, a spheroid, an organoid, or any combination thereof.

[0099] In some instances, the cells in the cell aggregates are all of the same type. For example, they can all be brain cells, muscle cells, or heart cells. In other instances, the cells in the multicellular aggregates are all from the same lineage, for example, all hematopoietic precursor cells. In some instances, the cells are stem cells, such as neural stem cells or embryonic stem cells.

[0100] Thus, in one example, the multicellular aggregate comprises homogeneous or heterogeneous cell types."Cell growth"refers to the division or proliferation of seeded cells.

[0101] "Seeding" cells refers to applying an initial cell population to a cell culture medium or support. "Seeded cells" are cells that are initially applied to a support.

[0102] The term "detached" or "detached" with reference to cells on a support means that the cells are separated from the support such that they are no longer anchored or adhered to the support.

[0103] The term "detachment" or "detached" with respect to cell aggregates refers to the separation of cells from the cell aggregate such that they are no longer anchored or adhered to the cell aggregate.

[0104] As used herein, "application frequency" refers to the number of times an agent, in particular a metalloprotease (such as collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) is added to the cell culture medium before and / or during the cell culture period.

[0105] The "logarithmic growth phase" is the logarithmic or exponential phase of cell growth, in which cells are actively proliferating and cell density is increasing. The logarithmic phase typically follows the lag phase and precedes the stationary phase, in which growth slows or stops.

[0106] As used herein, the term "metalloprotease" refers to a protease having one or more metal ions at the binding / active site. Examples of metalloproteases include collagenase and / or dispase.

[0107] The term "cysteine protease" is intended to describe proteases that have a highly reactive thiol group on 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, a cysteine protease may be of the CA family (sometimes also referred to as papain-like proteases). Papain-like proteases share a common catalytic binary active site characterized by a cysteine amino acid residue that acts as a nucleophile. Suitably, the cysteine protease of the CA family may be selected from the group consisting of: ficin, papain, bromelain, cathepsin K, and calpain. More suitably, the cysteine protease of the CA family may be ficin, papain, and / or bromelain. More suitably, the cysteine protease of the CA family may be ficin and / or papain. The term "CA family" is based on the MEROPS classification scheme (https: / / www.ebi.ac.uk / merops / ).

[0108] Suitably, papain can be formulated as Papain is a powdered preparation of purified papain standardized with maltodextrin. Papain is an enzyme extracted from the unripe green fruit of the papaya tree.

[0109] Ficin (also known as ficain) is a proteolytic enzyme typically extracted from the latex sap of the stems, leaves, and unripe fruit of the American wild fig tree, Ficus insipida.

[0110] The invention provides a kind of adherent cell culture method, wherein during the proliferation (or growth) phase, cells are detached (for example, detached from a support or each other, for example, detached from cell aggregates), so that the stationary phase can be delayed, and cell yield can be increased. The present invention is based on providing metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) of detached cells in a cell culture medium during the growth phase. Therefore, the invention provides a method for the continuous growth and detachment of adherent cells. In a defined cell culture phase comprising all or part of a logarithmic phase, under conditions suitable for cell growth / division, cells are maintained in a cell culture medium and metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin). In the cell culture period defined, cells can be continuously detached, which means that in the method of the present invention, the metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) present in the cell culture medium continuously detach the cells from the support and / or from other adherent cells (e.g., from cell aggregates), and therefore do not reach a stationary phase of growth. Surprisingly, the present inventors have found that other types of proteases, such as serine proteases (e.g., trypsin), do not have the same effect under the background of continuous cell detachment. Without wishing to be bound by this hypothesis, the present inventors believe that this is because metalloproteinases and cysteine proteases have different mechanisms of action compared to serine proteases. Specifically, serine proteases can transmit intracellular signals, while metalloproteinases and cysteine proteases act on the extracellular matrix, rather than together with cells / acting on cells, and do not directly trigger any intracellular response.

[0111] Continuous cell detachment may be particularly useful in the context of continuous bioprocessing systems. In contrast to traditional cell culture systems, in which cells are harvested in bulk after entering stationary phase, continuous bioprocessing systems allow adherent cells to be cultured and harvested with minimal human intervention and increased productivity per unit volume.

[0112] In traditional cell culture systems, during the harvesting step, an enzyme, such as trypsin, is added to the cell culture vessel at the end of the process to detach the cells and allow them to be harvested from the culture vessel.

[0113] In contrast, in continuous bioprocessing, cell detachment agents (such as metalloproteases and / or cysteine proteases) can be supplied to the cell culture all the time (not just at the end of the process) to allow a small proportion of cells to detach, allowing other cells to divide. When a small proportion of cells detach, the detachment of the cells therefore occurs gradually over time rather than as a discrete (batch detachment) event.

[0114] It will be understood that in the context of the present disclosure, the supply of cell detaching agents, such as metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) to the cell culture medium can be continuous or can be semi-continuous (e.g., intermittent). In some examples of the invention, the cell detaching agent can be supplied continuously during the culture of adherent cells. In other examples of the invention, the cell detaching agent can be supplied at specific time intervals during the culture of adherent cells. In either example, the amount of cell detaching agent supplied can be increased or decreased in response to the measured cell density or confluence. In either example, the cell detaching agent is supplied throughout the culture process (i.e., not just in a single discrete (batch detachment) event, such as a harvesting event or step at the end of the culture period).

[0115] The methods of the present invention have numerous advantages. For example, by maintaining cells in the logarithmic growth phase for a longer period of time, the methods increase the yield of cells in cell culture. Furthermore, the methods offer the advantage of reducing the amount of resources required to produce a desired yield, thereby reducing the amount of resources or carbon footprint.

[0116] In one aspect of the present invention, a method for culturing an adherent cell population is provided, the method comprising:

[0117] (i) culturing the cell population in a cell culture medium comprising a metalloprotease (such as collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) for a culture period that comprises all or a portion of a logarithmic growth phase of the cell population, wherein the metalloprotease (such as collagenase and / or dispase) and / or the cysteine protease (such as papain and / or ficin) continuously detaches a portion of the cells in the cell population.

[0118] In another aspect of the present invention, a cell culture method for an adherent cell population is provided, the method comprising:

[0119] (i) culturing the cell population in a cell culture medium comprising a metalloprotease (such as collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) for a culture period of at least 24 hours, wherein the metalloprotease (such as collagenase and / or dispase) and / or the cysteine protease (such as papain and / or ficin) continuously detaches a portion of the cells in the cell population.

[0120] The method of the invention may comprise one or more steps prior to step i). The method may comprise, for example, obtaining a cell population from a suitable source.

[0121] The present invention is suitable for the growth of any adherent cells. Anchorage-dependent cells are generally derived from multicellular organisms. Suitably, adherent cells can be mammalian cells, such as humans, mice, rats, rabbits, dogs, cats, cattle, pigs, chickens, goats, horses, etc. Mammalian cells can be derived from any suitable tissue, such as adrenal glands, bladders, blood vessels, bones, bone marrow, brain, cartilage, cervix uteri, cornea, endometrium, esophagus, gastrointestinal tract, immune system (e.g., T lymphocytes, B lymphocytes, leukocytes, macrophages and dendritic cells), liver, lungs, lymphatic vessels, muscles (e.g., myocardium), nerves, ovaries, pancreas (e.g., islet cells), pituitary gland, prostate, kidney, saliva, skin, tendons, testicles and thyroid gland. In some embodiments, cells are mammalian cells (e.g., humans). Adherent cells can be non-mammalian cells, such as insect cells, bird cells or fish cells. Cells can be prokaryotic cells, such as fungal cells or bacterial cells. Adherent cells can be plant cells. Suitably, adherent cells can be primary cells or immortalized cells. By way of example only, primary cells can be selected from the group consisting of: myocytes, cardiomyocytes, epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, osteoblasts, chondrocytes, adipocytes and mesenchymal stem cells. By way of example only, immortalized cells can 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 cells can be disease cells or disease model cells, such as cancer cells or cells 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.

[0122] Suitable cells are described elsewhere herein, and the skilled artisan will understand how to obtain such cell populations.For example, the method may comprise obtaining a suitable tissue and isolating cells therefrom.

[0123] Adherent cells are cultured as an adherent cell population. An adherent cell population can be cultured in a cell culture medium as cell aggregates (eg, wherein the adherent cells adhere to each other, rather than themselves to a support) or it can be cultured on a support.

[0124] A cell population produced in cell culture may form a layer on a support that is at least 50%, 60%, 70%, 80%, 90% or 100% confluent. Thus, the cell layer may be referred to as confluent. Suitably, the cell layer does not become overly confluent. Suitably, the cell layer is maintained at 80%-100% confluence during the cell culture period.

[0125] The layer of cells in contact with the support can be a monolayer of cells (meaning the layer is a single cell deep), or more than one layer of cells (two or more layers of cells deep). The cells can comprise one cell type, or two or more different cell types.

[0126] The support can be placed in a suitable growth chamber or container. The skilled person can select a suitable support for use in the present invention based on factors including the type of cells to be cultured and the desired outcome of the culture. If necessary, the support can be modified or adjusted to support or improve cell adhesion thereto.

[0127] Before use, the support is usually sterilized. Sterilization can be performed, for example, by gamma-irradiation, by autoclaving, by washing with alcohol or by treatment with ethylene oxide (ETO) gas.

[0128] Suitably, in the context of the present disclosure, a support can be any surface on which cells can grow. Suitably, the support is solid or semisolid. The support can be 2-dimensional (2D) or 3-dimensional (3D). Examples of 2D surfaces are the surface of a cover glass or a culture vessel (such as a tube, flask, dish or plate containing a plurality of wells). Examples of 3D surfaces are scaffolds, such as polystyrene scaffolds (e.g. Alvetex TM ) or a gel scaffold (e.g., a hydrogel).

[0129] The support can be a slide, a chip, a plate, a flask, a bottle, a film, a microstructure (comprising groove, hole or post), a pearl, a matrix, a resin, a gel, a film, a membrane, a porous plate, a double chamber plate or any other suitable form. The support can be any suitable shape or structure, comprising plane, tubular, curved, spherical, elliptical etc., comprising composite material (for example, to simulate macroscopic anatomical structure). The support can comprise a planar surface, and cell mass can be provided thereon. The support can be arranged or installed on a porous carrier (for example, porous membrane, mesh, inorganic grid, hydrogel or their combination), to provide structural support thereto. The support can be edible.

[0130] The support can be rigid or elastic. The support can be porous or impermeable. Materials that are not naturally porous can be made porous by methods available to the skilled artisan (e.g., sintering, etching, leaching, photolithography, or laser micromachining). Examples of porous supports can be gels or meshes.

[0131] In one example, the support comprises a material selected from the group consisting of: plastic, polymer, glass, and metal.

[0132] In one example, the support is selected from the group consisting of beads, microcarriers, microfluidic chips, silicon chips, microscope slides, microplate wells, matrices, resins, biochips, multiwell plates, gels, films, and membranes.

[0133] Should be understood that all or part of of support can be made by the material that promotes cell adhesion or with the material treatment that promotes cell adhesion.The material that promotes cell adhesion can be selected from the group consisting of: polyester, polypropylene, polyalkylene, polychlorofluoroethylene, polyvinyl chloride, polyvinyl fluoride resin, polystyrene, polysulfone, polyurethane, polyethylene terephthalate, cellulose, glass fiber, ceramic particles, matrigel, extracellular matrix components, collagen, poly-L lactic acid, dextran, inert metal fiber, silicon dioxide, natron glass, borosilicate glass, chitosan or plant sponge. Suitably, cellulose can be cellulose acetate. Extracellular matrix components can be one or more in fibronectin, vitronectin, chondrocyte adhesion protein or laminin. Suitably, adhesive material is electrostatic. Suitably, adhesive material can be coated with collagen or gelatin. Suitably, the adhesive material may comprise a peptide amphiphile (PA), such as that described in Miotto et al, Developing a Continuous Bioprocessing Approach to Stromal Manufacture, ACS Applied Materials & Interfaces 2017 9(47), 41131-41142. Adherent cells may attach to the surface via an anchoring substrate such as an integrin or other cell receptor. The support may comprise a non-adhesive portion.

[0134] The method of the present invention can be carried out in a growth chamber. As will be clear to those skilled in the art, a "growth chamber" refers to any suitable chamber or container suitable for adherent cell growth. Therefore, a "growth chamber" can have any suitable type of surface and any suitable type of geometry. Suitable growth chambers are well known in the art and include, but are not limited to, microcarriers, cell culture plates, cell culture containers, reaction vessels, and bioreactors. Therefore, suitable growth chambers can include, but are not limited to, hollow fibers, stirred tanks, air lift chambers, bubble towers, fluidized bed reaction vessels / bioreactors, or flexible bags. Therefore, it is apparent that any suitable growth chamber (e.g., bioreactor) can also be used.

[0135] Prior to step i), the method of the present invention may comprise one or more of culturing, maintaining, passaging, separating and / or isolating the cell population. When the cells are cultured or maintained in a cell culture medium prior to step i), this may be performed in the absence of metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin).

[0136] 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 does not require supplementation for culturing cells; or it may be an incomplete formulation, i.e., a cell culture medium that requires supplementation; or it may be a culture medium that can supplement an incomplete formulation; or, in the case of a complete formulation, may improve the culture or culturing results. Various cell culture media are known to those of ordinary skill in the art, who will also understand that the type of cell to be cultured may determine the type of culture medium to be used. Suitably, a cell culture medium is selected that does not substantially affect the activity of the selected metalloproteinase (such as collagenase and / or dispase) and / or cysteine protease (such as papain and / or ficin).

[0137] By way of example only 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, Mesencult TM Basal medium, minimum essential medium (MEM), basal medium Eagle (BME), Ham's F-10, alpha minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM) and Iscove's modified Dulbecco's medium (IMDM), or any combination thereof. Other culture media commercially available (e.g., from Thermo Fisher Scientific, Waltham, Massachusetts) or otherwise known in the art can be equally used in the context of the present disclosure. Again, by way of example only, the culture medium can be selected from the group consisting of: 293SFM, CD-CHO medium, VP SFM, BGJb medium, Brinster's BMOC-3 medium, cell culture freezing medium, CMRL medium, EHAA medium, eRDF medium, Fischer's medium, Gamborg's B-5 medium, supplemented with GLUTAMAX TMmedium, Grace's insect cell medium, HEPES-buffered medium, Richter's modified MEM, IPL-41 insect cell medium, McCoy's 5A medium, MCDB 131 medium, medium 199, modified Eagle's medium (MEM), medium NCTC-109, Schneider's Drosophila medium, TC-100 insect medium, Waymouth's MB 752 / 1 medium, William's medium E, protein-free hybridoma medium II (PFHMII), AIM V medium, Keratinocyte SFM, defined Keratinocyte SFM, SFM, Complete methylcellulose medium, HepatoZYME-SFM, Neurobasal TM Culture medium, Neurobasal-A medium, Hibernate TM A medium, Hibernate E medium, Endothelial SFM, Human Endothelial SFM, Hybridoma SFM, PFHMII, Sf 900 medium, Sf 900II SFM, EXPRESS Culture medium, CHO-S-SFM, AMINOMAX-II complete culture medium, AMINOMAX-C100 complete culture medium, AMINOMAX-C140 basal culture medium, PUB-MAX TM Karyotyping Medium, KARYOMAX Bone Marrow Karyotyping Medium, and KNOCKOUT D-MEM, or any combination thereof.

[0138] Suitably, the cell culture medium may be serum-free. Suitably, the cell culture medium may be glucose-free.

[0139] Alternatively, the cell culture medium may contain serum. Suitable types and amounts of serum are known, for example 1% FBS may be used.

[0140] Metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) may be described as "exogenous metalloproteases and / or exogenous cysteine proteases".

[0141] As used herein, the term "exogenous" refers to a protease (particularly a metalloprotease and / or a cysteine protease) that is added to a cell culture medium (i.e., the cell culture medium is already supplemented with said enzyme). Thus, "exogenous" refers to a protease (particularly a metalloprotease and / or a cysteine protease) that is not expressed by the cell or at least not expressed in the amount present in the cell culture medium. In other words, the term "exogenous" refers to a protease (particularly a metalloprotease and / or a cysteine protease) that comes from an external source (it is not naturally produced in that amount by the cells in the cell culture medium).

[0142] For example, as used herein, the term "exogenous collagenase" (or "exogenous dispase") refers to a collagenase (or dispase) that is added to a cell culture medium (i.e., the cell culture medium has been supplemented with collagenase (or dispase)). It refers to a collagenase (or dispase) that is not expressed by the cells present in the cell culture medium (herein, metalloproteinases expressed by cells present in the cell culture will be considered endogenous collagenases (or dispase)). Thus, it is a collagenase (or dispase, or cysteine protease, such as papain and / or ficin) that comes from an external source (it is not naturally produced in that amount by the cells in the cell culture medium). For the avoidance of doubt, the cells in the cell culture medium may also be capable of producing these proteases, however, the exogenous collagenases (or dispase) referred to herein are those that are added to the cell culture medium from an external source.

[0143] Exogenous metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) may also be referred to herein as cell culture medium supplements or supplemented metalloproteases (such as collagenase and / or dispase) and / or supplemented cysteine proteases (such as papain and / or ficin).

[0144] Suitably, the cell culture medium may contain one or more additional agents as needed. These agents may be selected from, but not limited to, the group consisting of: antibiotics, buffers, growth factors, hormones, nutritional supplements, indicators, and essential metals and minerals.

[0145] In the next step before step i), the cells can be seeded on a support or incubated into a growth chamber (e.g., to grow as cell aggregates). Suitably, after the support is incubated with the cell culture medium, the cells are seeded on the support, or the cells are incubated directly into the cell culture medium in the growth chamber. Methods of incubating cells into cell culture medium are well known and can be adjusted according to the cell type. In addition, methods of cell seeding are known and available to those of ordinary skill in the art and can be adjusted according to the cell type and support. Two or more supports can be seeded simultaneously. The initial cell seeding density must be effective while allowing optimal cell proliferation within the support. The number of cells to be seeded further depends on the porosity of the scaffold material and any liquid absorption capacity. For porous supports, the greater the porosity, the greater the number of cells that can be seeded. In some embodiments, the number of cells per gram of support (dry weight) is between 2×10 6 to 50×10 6 Furthermore, the porosity of the scaffold and the internal organization of the support fibers contribute to the retention of cells in and on the support. The method may comprise performing more than one sequential seeding step on the support.

[0146] Before the cell culture medium is applied to the cell culture, a metalloproteinase (such as collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) can be provided in the cell culture medium. Therefore, a cell culture medium comprising a metalloproteinase (such as collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) can be added to the support or growth chamber itself. Alternatively, a metalloproteinase (such as collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) can be provided to the cell culture medium alone. Suitably, before the incubation period begins, a metalloproteinase (such as collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) can be added to the cell culture medium. Therefore, a metalloproteinase (such as collagenase and / or dispase) and / or a cysteine protease (such as papain and / or ficin) can be added to the cell culture medium during the lag phase of cell growth. Alternatively or additionally, metalloproteases (such as collagenases and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to the cell culture at the end of the logarithmic phase of cell growth or when the cells reach a confluence of at least 50%, at least 60%, at least 70%, etc. In some examples, metalloproteases (such as collagenases and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to the cell culture when the cells reach a confluence of at least 80% or 85%.

[0147] During the incubation period, additional metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to the cell culture. During the incubation period, one or more additional metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to the cell culture. For example, during the cell culture period, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more additional metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to the cell culture. Supplementary metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to the cell culture at regular or irregular intervals. In some embodiments, the metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) are administered at regular intervals. For example, conventional intervals can be per 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 extra metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be referred to as "pulses". If supplementary metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) are added at irregular intervals, these intervals can depend on factors such as the concentration of metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) in the growth rate of cells, desorption rate, the degree of confluence on the support, and / or the cell culture medium. Supplemental metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be provided to cell cultures where the detachment rate is lower than the growth rate, e.g., 10%, 20%, 30%, 40% or 50% or more lower.

[0148] Supplementary metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added together with the cell culture medium. In such an embodiment, a certain amount of existing cell culture medium can be removed to maintain the desired concentration of metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin). Suitably, removing or adding cell culture medium is not equivalent to washing cells, and the growth phase of cells can not be changed when being in the logarithmic phase. In the cell culture phase, the cell culture medium is not completely removed, and washing medium is not added to the cell culture.

[0149] The cell culture is maintained under appropriate cell culture conditions for at least the duration of the cell culture period. Suitable cell culture conditions for different cell types are known to those of ordinary skill in the art. Typical growth conditions are 37°C, 95% relative humidity, and 5% CO availability. The pH can be maintained between 7 and 7.4.

[0150] Cell cultures can be maintained under static or dynamic conditions. In a dynamic system, motion is applied to the cell culture (e.g., by placing it on a rocker) to allow movement of the cell culture medium. In a static system, no mechanism is provided for the movement of the cell culture medium.

[0151] The cell culture period can be defined as all or part of the logarithmic growth phase comprising a cell mass, or can be defined as at least 24 hours. The culture period can be at least 24 hours, and can comprise all or part of the logarithmic growth phase of a cell mass. The logarithmic phase can be any period between the lag phase and the stationary phase, wherein the cells in the culture are actively propagating. Suitably, the culture period comprises all or part of the logarithmic phase, suitably at least 40%, 50%, 60%, 70%, 80% or at least 90% of the logarithmic phase. Suitably, the culture period does not comprise all or part of the lag phase or the stationary phase. Any suitable method can be used, for example visual or analytical methods (such as cell counting, DAPI staining etc.) to determine the growth phase of cell culture.

[0152] Suitably, cell culture can 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 longer. Suitably, cell culture can continue at least about 84 hours, at least about 96 hours (about 4 days), 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 longer. Suitably, cell culture can be about 1 day to about 9 days, for example, about 2 days to about 8 days or about 3 days to about 7 days.

[0153] Suitably, the cells can be cultured for 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 longer. Suitably, the cells can be cultured for at least about 40 days.

[0154] Suitably, the cells can be cultured for 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 longer. Suitably, the cells can be cultured for at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, or longer.

[0155] In the method for the present invention, suitably, cell (for example, on support) does not reach confluence during the cell culture phase, or suitably does not become excessive confluence.Suitably, method of the present invention enables cell to maintain about 50%-100%, more suitably 60%-95%, more suitably 80%-95% or any scope or integer therebetween during the cell culture phase, for example, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% confluence.Suitably, confluence maintains lower than 100% during the cell culture phase.By using metalloproteinase (such as collagenase and / or dispase) and / or cysteine protease (such as papain and / or ficin) in cell culture medium during the cell culture phase, confluence can be maintained at level as described herein, this allows along with cell proliferation and colony growth, the continuous detachment of cell (for example, from support or from other adherent cells, for example, from cell aggregate).

[0156] In the methods of the present invention, metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) continuously detach a portion of the cells in the cell population. In this case, "continuously detaching a portion of the cells in the cell population" refers to continuously removing a portion of the adherent cells in the cell population (e.g., removing a portion of the adherent cell population on a support or removing a portion of the adherent cell population in an aggregate). Thus, detachment occurs gradually over time rather than as a discrete (batch detachment) event.

[0157] As will be clear to those skilled in the art, continuous detachment occurs over a period of time (in the incubation period, typically more than 24 hours). Therefore, metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) cause a portion of cells in the cell mass to continuously detach during the incubation period. Typically, the method of the present invention is included in the detachment of adherent cells (using dispase and / or collagenase) from the cell mass when the cell mass undergoes growth phase (typically when the cell mass is in the logarithmic phase). Therefore, continuous detachment can be used to maintain the cell mass in a confluence or density that enables the cell mass to maintain the logarithmic growth phase (or at least avoid the stationary phase and lag phase of cell growth) during the incubation period. Therefore, during step (i), the method of the present invention typically maintains the adherent cell mass at a confluence of 40% to 95% (for example, when adhering to a support). Optimal confluence is discussed elsewhere herein and can be easily determined by those skilled in the art.

[0158] Thus, metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can continuously detach a portion (e.g., at least 5%, but not more than 60%) of the cells in the cell population during the culture period.

[0159] Thus, metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can continuously detach a portion (e.g., at least 0.5%, but not more than 7% / hour) of the cells in the cell population during the culture period.

[0160] In the methods of the present invention, continuous detachment can maintain the cell confluence or density of the adherent cell population during the culture period (in other words, in some examples, the cell confluence or density can increase or decrease by no more than 40% during the culture period).

[0161] In the methods of the present invention, continuous detachment can maintain the cell confluence or density of the adherent cell population during the culture period (in other words, in some examples, the cell confluence or density does not increase or decrease by an average of more than 40% per week during the culture period).

[0162] As will be clear to those skilled in the art, "continuous detachment" does not include batch detachment (a term well known in the art). In the context of metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin), batch detachment can refer to detachment of at least 60% of the cells in a population of adherent cells within 30 minutes at 37°C. Therefore, step (i) of the method of the present invention does not include batch detachment of cells in a cell population.

[0163] Suitably, in the incubation period, the desorption rate exceeds the proliferation rate. Suitably, metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) are provided in the cell culture medium at a concentration in the range of 0.3:1 to 1:3 with the ratio of the desorption rate (for example, from support or from other adherent cells, for example, from cell aggregates) and the growth rate of the cell. In other words, the desorption rate can be one-third or more of the proliferation rate. The proliferation rate can be up to 3 times of the desorption rate. Desorption rate and proliferation rate can be determined by, for example, imaging, bioreactor sensor / probe, analytical method, and all of these are well known in the art.

[0164] Suitably, the detached cell or a part of the detached cell can be gathered in the crops at least once during the cell culture phase. Suitably, the detached cell or a part of the detached cell can be gathered in the crops more than once during the cell culture phase. The harvesting of the detached cell can be carried out per 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. From culture, the suitable method of removing the detached cell comprises using dead end filtration or cross flow / tangential fluid flow filtration, centrifuge, acoustic separation or a system based on microfluidics. Alternatively, the detached cell is not gathered in the crops, and can be retained in the cell culture after the cell culture phase.

[0165] Before or after step i) of the method of the present invention, the cells may be passaged one or more times. Thus, the method may comprise removing cells from the cell culture and placing the cells on a new support or growth chamber. Suitably, the cells are a cell population detached by the method of the present invention.

[0166] The methods of the present invention may additionally include monitoring or determining the viability of cells in the cell culture. Cell viability can be measured by measuring cell proliferation or metabolic activity. Other methods include flow cytometry and immunohistochemistry. Measurement of cell viability can be performed after the incubation period in step i).

[0167] Suitably, in the methods of the invention, the metalloproteinase (such as collagenase and / or dispase) and / or cysteine protease (such as papain and / or ficin) has no substantial detrimental effect on 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 case, a substantial detrimental effect may be considered to be a 10% reduction in cell viability over the culture period, compared to the cell viability observed in the absence of the metalloproteinase (such as collagenase and / or dispase) and / or cysteine protease (such as papain and / or ficin).

[0168] Method of the present invention can additionally include monitoring or determining the confluence (for example, on support) of cell.Can use chemical dye (for example, thymidine, alamar blue (alamar blue), XTT or other dyestuff available in this area), qualitative visual measurement state or use image processing method (comprising that for example Olympus CKX53 cultivates microscope, CKX-CCSW confluence checker software and air fraction output) to measure cell confluence.As described in example below, Cytation1, IoLight, Jiusion USB digital microscope and ImageJ can also be used to image and analyze cell.When method shows that cell is more confluent than expectation, can add extra metalloproteinase (such as collagenase and / or dispase) and / or cysteine protease (such as papain and / or ficin) to increase the speed that cell is detached from support in cell culture medium.Can consider the total concentration of metalloproteinase (such as collagenase and / or dispase) and / or cysteine protease (such as papain and / or ficin) to remain in the optimal limit of cell type.

[0169] Cell confluence can also be referred to as the density of adherent cells (e.g., on a support). Cell density can be determined using, for example, microscopy, acoustic resonance density determination, laser induced fluorescence, fluorescence microscopy, capacitance impedance, turbidity, biomass dielectric constant probes, Raman probes, and cell counters (e.g., CCD imaging using trypan blue). Cell density can also be used to determine the rate at which cells detach from cell aggregates, as described elsewhere herein.

[0170] The methods of the present invention may comprise monitoring or determining the growth phase of cells in culture. This may be done using visual means, such as examining cell shape.

[0171] Any suitable cell imaging method can be used, for example, to image or otherwise analyze cells, such as fluorometers, luminometers, cameras, microscopes, plate readers, cell analyzers, and confocal imaging systems.

[0172] The method of the present invention may comprise repeating step i) two or more times.

[0173] Suitably, the incubation period in step i) does not include a step of washing the cells (e.g., washing the support containing the cells). A washing step may be included after step i). Washing may be performed using any suitable method, depending on the choice of cells (and, for example, the choice of support). Washing may include aspirating the cell culture medium and placing the cells in a physiological buffer.

[0174] The methods of the invention may comprise counting cells.

[0175] Suitably, substantially all cells of the cell population do not differentiate during the cell culture period.Thus, substantially all cells of the cell population will not show signs of differentiation, such as differentiation markers or changes in cell shape or morphology.

[0176] The methods of the present invention may further comprise culturing the cells under conditions suitable to allow differentiation.

[0177] The method of the present invention may further comprise cryopreserving the harvested cells.

[0178] Any suitable source of metalloproteinases (such as collagenases and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be used in the present invention. Suitably, the metalloproteinases (such as collagenases and / or dispase) and / or cysteine proteases (such as papain and / or ficin) are compatible with the cell culture medium. Suitably, the metalloproteinases (such as collagenases and / or dispase) and / or cysteine proteases (such as papain and / or ficin) are capable of detaching cells without substantially affecting cell growth or viability.

[0179] The metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) may be pure or may contain trace amounts (eg, 15% or less, or 10% or less) of other proteins or enzymes.

[0180] In one example, type I collagenase can contain other enzymes, such as tyrosine kinase, clostripain and trypsin. Suitably, type I collagenase can have more than 125 U / mg of collagenase; more than 200 U / mg of tyrosine kinase; less than 4 U / mg of clostripain and less than 0.5 U / mg of trypsin activity.

[0181] When the metalloprotease is a collagenase, it may be a collagenase obtained from Clostridium histolyticum and also used in human medicine. However, it may also be isolated from other Clostridium bacteria or tissues (e.g., Merck Index No. 2477).

[0182] A suitable collagenase may be Gibco isolated from Clostridium histolyticum. TM Type I collagenase (product code 11500536, from Fisher Scientific), which contains average levels of collagenase, tyrosinase, clostripain, and trypsin activities, was used in the following examples.

[0183] Many FDA-approved collagenases are known in the art and available. For example, these include Santyl (Smith & Nephew); cellulite (Qwo, Endo International) and Xiaflex (Endo International).

[0184] Suitably, a single metalloprotease (i.e., collagenase or dispase) or cysteine protease (i.e., papain or ficin) can be used in the cell culture during the single cell culture period. Alternatively, a combination of two or more enzymes (i.e., metalloprotease or cysteine protease) can be provided in the cell culture during the single cell culture period. As an example only, a metalloprotease (i.e., collagenase or dispase) and a cysteine protease (i.e., papain or ficin) can be used in the cell culture during the single cell culture period, or a combination of two or more metalloproteases (such as collagenase and / or dispase) and / or cysteine protease (such as papain and / or ficin) can be used in the cell culture during the single cell culture period. A combination of metalloprotease and cysteine protease can also be used in the cell culture during the single cell culture period.

[0185] As will be clear to those skilled in the art, any suitable concentration or amount of metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be used, provided that the metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) continuously desorb a portion of the cells in the cell mass during the incubation period. Those skilled in the art can easily determine suitable concentrations and amounts based on the disclosure herein and common general knowledge thereof. The exact amount to be used can depend on many factors, such as the desired desorption rate, cell culture medium, whether static conditions or dynamic conditions are used, etc. Some non-limiting examples are provided herein. For example, it is shown that under serum-free conditions, in a planar system, static culture requires a higher concentration of collagenase compared to dynamic culture. When using microcarriers, even higher concentrations are required. When serum is present in the cell culture medium, a further increase in concentration is also observed (planar static-steady-state continuous desorption is achieved using 9 to 20 U / mL of type I collagenase; compared to using 5 to 10 U / mL to achieve planar dynamic-steady-state continuous desorption; batch desorption requires 4200 U / mL (20 mg / mL) - data not shown). Therefore, a suitable concentration can be determined by those skilled in the art. Suitably, when collagenase is used, it can be provided in cell culture at 500 U / mL or less. Suitably, it can be provided in cell culture at 215 U / mL or less. Suitably, collagenase can be provided in cell culture at 120 U / mL or less. Suitably, collagenase can be provided in cell culture at 110 U / mL or less, or 50 U / mL or less.

[0186] Suitably, when the collagenase is type I collagenase, it may be provided in the cell culture at 215 U / mL or less. Suitably, type I collagenase may be provided in the cell culture at 120 U / mL or less. Suitably, type I collagenase may be provided in the cell culture at 110 U / mL or less. Suitably, type I collagenase may be provided in the cell culture at 80 U / mL or less. Suitably, type I collagenase may be provided in the cell culture at 50 U / mL or less. Suitably, type I collagenase may be provided in the cell culture at 20 U / mL or less. Suitably, type I collagenase may be provided in the cell culture at 10 U / mL or less. Suitably, type I collagenase may be provided in the cell culture at 5 U / mL or less. Suitably, type I collagenase may be provided in the cell culture at 3 U / mL or less. Suitably, collagenase type I can be provided in cell culture with 2U / mL or lower. Suitably, collagenase type I can be provided in cell culture with 1U / mL or lower or any scope formed by any upper limit or lower limit of above-mentioned scope or any integer therebetween or at least, no more than, lower than or higher than any value of above-mentioned scope. In each case in these situations, the lower limit of scope can be 0.005U / mL, for example 0.008U / mL. For example, when collagenase is collagenase type I, it can be provided in cell culture with a concentration of 0.008U / mL to 20U / mL. For example, when collagenase is collagenase type I, it can be provided in cell culture with a concentration of 5U / mL to 20U / mL or with a concentration of 9U / mL to 20U / mL. For example, when collagenase is collagenase type I, it can be provided in cell culture with a concentration of 0.008U / mL to 10U / mL. For example, when collagenase is type I collagenase, it can be provided in cell culture at a concentration of 5U / mL to 10U / mL or a concentration of 0.008U / mL to 5U / mL. Suitably, when collagenase is type VII collagenase, it can be provided in cell culture at 500U / mL or lower. Suitably, type VII collagenase is provided in cell culture at 300U / mL or lower. Suitably, type VII collagenase is provided in cell culture at 200U / mL or lower. Suitably, type VII collagenase is provided in cell culture at 150U / mL or lower. Suitably, type VII collagenase can be provided in cell culture at 120U / mL or lower. Suitably, type VII collagenase can be provided in cell culture at 110U / mL or lower. In each of these cases, the lower limit of the scope can be 50U / mL. For example, when the collagenase is type VII collagenase, it can be provided in the cell culture at a concentration of 50 U / mL to 300 U / mL.For example, when collagenase is collagenase type VII, it can be provided in cell culture at a concentration of 50U / mL to 200U / mL or a concentration of 50U / mL to 150U / mL. For example, when collagenase is collagenase type VII, it can be provided in cell culture at a concentration of 50U / mL to 120U / mL. For example, when collagenase is collagenase type VII, it can be provided in cell culture at a concentration of 50U / mL to 110U / mL.

[0187] Suitably, when enzyme is dispase, it can be provided in cell culture medium with 13U / mL or lower. Suitably, dispase exists in cell culture medium with 6.5U / mL or lower. Suitably, dispase exists in cell culture medium with 3U / mL or lower. Suitably, dispase exists in cell culture medium with 2U / mL or lower or 1U / mL or lower. Suitably, dispase exists in cell culture medium with 0.8U / mL or lower or 0.5U / mL or lower. In each of these cases, the lower limit of the scope can be 0.0005U / mL, for example 0.0008U / mL. For example, dispase can be provided in cell culture with a concentration of 0.0008U / mL to 3U / mL. For example, dispase can be provided in cell culture with a concentration of 0.0008U / mL to 2U / mL or 0.0008U / mL to 1U / mL. For example, the dispase can be provided in the cell culture at a concentration of 0.0008 U / mL to 0.8 U / mL. For example, the dispase can be provided in the cell culture at a concentration of 0.0008 U / mL to 0.5 U / mL.

[0188] Enzyme unit (U) is the measurement of enzyme catalytic activity, and enzyme unit (U) is the amount of the enzyme that catalyzes 1 micromole of substrate conversion per minute under specific conditions. Alternatively, enzyme activity can be represented by Katal (katal) (enzyme activity of converting 1 mole of substrate per second under specific conditions). For collagenase, enzyme activity unit can be collagenase degradation unit (CDU or Mandel (Mandl)), where one CDU catalyzes the hydrolysis (Lockhardt et al J. Stem Cell Res Ther, 5: 321 (2015)) of 1 micromole of L-leucine equivalent in collagen at 37 DEG C, pH 7.4 within 5 hours. Specific conditions according to the present invention are those defined in an example, such as static or dynamic serum-free culture on glass microcarriers. Technicians can determine the enzyme unit equivalent of different conditions or enzyme by measuring the enzyme activity under different conditions and regulating the amount of enzyme to reach the desired enzyme activity level under different conditions. For commercially available enzymes, the amount of enzyme measured as weight / volume (e.g., mg / mL) in the cell culture can be determined, for example, by using the enzyme % and enzyme activity units provided by the distributor. Therefore, for any commercially available enzyme, the technical staff can determine the amount of product required for the enzyme unit to provide the desired enzyme. For example, for type I collagenase, based on the weight of the product used in the ratio of protease and the cell culture in the product, the enzyme unit of 110U / mL is equivalent to 0.5mg / mL. Similarly, the enzyme unit of 215U / mL of type I collagenase used herein is equivalent to 1mg / mL. For type I dispase used herein, the enzyme unit of 6.5U / mL is equivalent to 0.5mg / mL, and the enzyme unit of 13U / mL is equivalent to 1mg / mL.

[0189] When representing with the concentration of enzyme, metalloproteinase (for example collagenase and / or dispase) can be with 1mg / mL or lower or suitably with 0.5mg / mL or lower provision in cell culture medium.Suitably, protease (for example collagenase and / or dispase) can be with about 1mg / mL, 0.9mg / mL, 0.8mg / mL, 0.7mg / mL, 0.6mg / mL, 0.5mg / mL, 0.4mg / mL, 0.3mg / mL, 0.2mg / mL, 0.1mg / mL or lower, more suitably with 0.09mg / mL, 0.08mg / mL, 0.07mg / mL, 0.06mg / mL, 0.05mg / mL, 0.04mg / mL, 0.03mg / mL, 0.02mg / mL, 0.01mg / mL, 0.005mg / mL, 0.001mg / mL or 0.0005mg / mL or lower provision. Suitably, the metalloprotease (e.g., collagenase and / or dispase) can be provided in the cell culture medium at a concentration 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 / L, or any range formed by any upper or lower limit of the aforementioned ranges, or any integer therebetween, or at least, no more than, less than, or greater than any value of the aforementioned ranges.

[0190] Suitably, the exogenous cysteine protease (e.g., papain and / or ficin) may be provided at 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 or less, more suitably 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 or less.

[0191] Suitably, ficin can be provided in a cell culture medium at a concentration 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 / L, or any range formed by any upper or lower limit of the above ranges, or any integer therebetween, or at least, not more than, less than, or greater than any value of the above ranges. In another example, ficin can be provided in a cell culture medium at a concentration of about 0.005 mg / mL to 0.1 mg / mL, for example, about 0.01 mg / mL to about 0.05 mg / mL.

[0192] Suitably, ficin can be provided in the cell culture medium at a concentration of about 0.0001 to about 0.1 B A A U / mL, such as about 0.0005 to about 0.01, about 0.001 to about 0.009, or such as about 0.002 to about 0.005 B A A U / mL. Suitably, ficin can be provided in the cell culture medium at a concentration of about 0.0033 B A A U / mL.

[0193] Suitably, papain can be in cell culture medium with about 0.0001mg / mL to 1mg / mL, 0.001mg / mL to 0.75mg / mL, 0.001mg / mL to 0.5mg / mL, 0.005mg / mL to 0.25mg / L or any scope formed by any upper limit or lower limit of above-mentioned scope or any integer therebetween or at least, be no more than, be lower than or provide higher than the concentration of any value of above-mentioned scope.In another example, papain can be in cell culture medium with about 0.0001mg / mL to 0.07mg / mL, for example, the concentration of about 0.001mg / mL to about 0.025mg / mL provides.Suitably, papain can be in cell culture medium with about 0.001 to about 10TU U / mL, for example, the concentration of about 0.05 to about 5, about 0.1 to about 2 or for example, about 0.5 to about 1TU U / mL provides. Suitably, papain may be provided in the cell culture medium at a concentration of about 0.79 TU U / mL.

[0194] The cells produced by the methods of the present invention can be used in a variety of research, diagnostic, drug screening, therapeutic, medical, industrial or food-based applications. For example, the cell cultures produced by the methods of the present invention can be used in the meat industry. Suitably, the methods of the present invention can include processing steps related to the use of the cells produced in the cell culture.

[0195] Batch desorption

[0196] In another aspect, the invention provides a method for desorbing cells in batches. The method comprises contacting the cells with a cysteine protease, such as the cysteine protease described above, wherein the cysteine protease desorbs the cells in batches. This aspect is based on the surprising discovery of the inventors that cysteine proteases can replace traditional serine proteases to desorb cells in batches. The cysteine proteases illustrated in this application are plant proteases, i.e., proteases naturally occurring in plants. It is believed that the use of such plant proteases in animal cell culture is novel and unexpected.

[0197] It will be appreciated that for such batch detachments, the concentration of the cysteine protease in the cell culture medium will be higher than that used for continuous cell detachment.

[0198] Suitably, in the case of batch cell detachment, the concentration of cysteine proteases (such as papain and / or ficin) may be higher than 1 mg / mL. For example, the concentration may be higher than 1.5 mg / mL, higher than 2 mg / mL, higher than 3 mg / mL, higher than 4 mg / mL, higher than 5 mg / mL, higher than 10 mg / mL, higher than 15 mg / mL, higher than 20 mg / mL, higher than 30 mg / mL, higher than 35 mg / mL.

[0199] Suitably, in the case of batch cell detachment, the concentration of the cysteine protease (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.

[0200] Suitably, in the case of batch cell detachment, the concentration of ficin may be higher than 0.1 B APA U / mL, such as higher than 0.3 B APA U / mL, or higher than 0.3 B APA U / mL. Suitably, the concentration of ficin may be from about 0.1 B APA U / mL to about 10 B APA U / mL, such as from about 0.3 B APA U / mL to about 0.5 B APA U / mL, or from about 0.39 B APA U / mL to about 6.3 B APA U / mL.

[0201] Suitably, in the context of batch cell detachment, the concentration of papain can be higher than 100 TU U / mL, for example higher than 200 TU U / mL. Suitably, the concentration of papain can be from about 100 TU U / mL to about 10,000 TU U / mL, for example from about 200 TU U / mL to about 5,000 TU U / mL or from about 200 TU U / mL to about 4,000 TU U / mL. Suitably, the concentration of papain can be from about 236 TU U / mL to about 3,772 TU U / mL.

[0202] Suitably, the cells can 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 longer. For example, the cells can be contacted with the cysteine protease for at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes or longer, such as at least 120 minutes or 150 minutes.

[0203] Suitably, the cells may be contacted with the cysteine protease for a period of about 10 to 200 minutes, such as about 20 to about 175 minutes, or about 30 to about 150 minutes. It will be clear to those skilled in the art that higher concentrations of the cysteine protease may allow the cells to be in contact with the enzyme for a shorter period of time.

[0204] Throughout the description and claims of this specification, the words "comprise" and "contain" and their variations mean "including but not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0205] Unless incompatible therewith, 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. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any disclosed method or process may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any foregoing embodiments. The invention extends to any new feature or any new combination of features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any new step or any new combination of steps of any disclosed method or process.

[0206] The reader's attention is directed to all papers and documents filed concurrently with or before 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.

[0207] Example

[0208] Example 1

[0209] Data were generated using C2C12 as a cell model grown in serum-free medium (DMEM F12 with 1% insulin-transferrin factor and 1 mM L-ascorbic acid).

[0210] 1.1. Collagenase can successfully detach C2C12 cells in bulk without changing their adhesion, proliferation, and differentiation potential. able.

[0211] Collagenase has been used previously for bulk detachment of certain types of adherent cells as an alternative to more common methods such as trypsin and TrypLE. To achieve bulk cell detachment in a short timeframe, a high concentration of collagenase was added and incubated at 37°C for 30 minutes (preheat the collagenase solution before addition). The concentration of type I collagenase used was 1180 U (or 472 U / mL; equivalent to 2.2 mg / mL). The detachment efficiency ( Figure 1 A) and vitality ( Figure 1 B) Collagenase supplementation showed comparable results to TrypLE. In addition, similar to control cells, collagenase-detached cells could reattach, proliferate, and differentiate ( Figure 1 CE).

[0212] 1.2. Collagenase can detach cells in a dose-dependent manner.

[0213] It was demonstrated that increasing amounts of collagenase resulted in increasing amounts of C2C12 cell detachment (Table 1).

[0214] Table 1: Quantification showing the increase in detached cell number using collagenase type I (units / cell) in a dose-dependent manner.

[0215]

[0216] This suggests that collagenase can surprisingly be used to control the rate of cell detachment. Furthermore, the detached cells were able to reattach and did not differentiate during the process ( Figure 2 ).

[0217] 1.3. Low concentrations of collagenase allow for continuous detachment of C2C12 cells.

[0218] It was demonstrated that administration of collagenase at lower concentrations allowed for continued cell detachment without affecting cell characteristics. This was studied in 4 different systems (see A to D below).

[0219] A. Continuous cell detachment in a planar system under static conditions.

[0220] It was demonstrated that supplementation with 1.75 U / mL (equivalent to 0.0081 mg / mL of type I collagenase used herein) allowed C2C21 cells to continue to grow and detach after 21 days of static culture in serum-free medium ( Figure 4Detached cells were collected every 24 hours and counted on days 1, 7, 9, 14, and 21. Confluence on the growth surface remained similar during the culture ( Figure 4 A), not grown to over-confluence. Every 48 hours, each surface with growing cells was moved to a different (new) well and fresh medium and collagenase were added. Over time, cells detached from the surface at similar levels ( Figure 4 B).

[0221] B. Continuous desorption in a planar system under dynamic conditions.

[0222] It was demonstrated that supplementation of 0.009 U / mL (equivalent to 0.00004 mg / mL (43.27 ng / mL) of type I collagenase used herein) allowed C2C12 cells to continue to grow and detach after 7 days of dynamic culture in serum-free medium ( Figure 5 Detached cells were collected at the end of the experiment, while growing cells were imaged daily ( Figure 5 A). At the end of the experiment, the number of attached cells harvested was similar to the number of cells seeded ( Figure 5 B). In addition, both attached and detached cells showed an initial undifferentiated phenotype at the end of 7 days of continuous culture under flow ( Figure 5 C&D).

[0223] C. Continuous desorption in a microcarrier system under static conditions.

[0224] It was demonstrated that supplementation with 4.5 U / mL type I collagenase enabled sustained growth and detachment of C2C12 cells after 27 days of static culture in serum-free medium.

[0225] During the culture period, the confluence of the growing cells on the microcarriers was maintained, and cell detachment was observed after collagenase supplementation starting on day 1 ( Figure 6 A)). Detached cells were collected every 24 h, and the average number of detached cells was consistent between the first and second weeks ( Figure 6 D), where approximately one-third of the initially seeded cells detached every 24 hours. The number of cells harvested from the microcarriers (on which they were previously grown) did increase from day 1, however, the cells did not overgrow on the microcarrier surface to cause aggregation ( Figure 6 E).

[0226] Furthermore, both (i) detached cells that detached during the process (yield) and (ii) adherent cells recovered from microcarriers at day 27 (grown on microcarriers) were able to reattach to tissue culture plastic after collection and displayed an initial undifferentiated phenotype ( Figure 7 A). In addition, the detached (yield) cells were able to reattach to fresh microcarriers in the presence of collagenase and undergo the same process again ( Figure 7 B).

[0227] D. Continuous desorption in microcarrier systems under dynamic conditions.

[0228] It was demonstrated that supplementation with 4.5 U / mL type I collagenase enabled sustained growth and detachment of C2C12 cells after dynamic culture in serum-free medium for 27 days.

[0229] In cultures supplemented with collagenase, cell detachment was consistently observed ( Figure 8 The detached cells were collected every 24 hours ( Figure 8 A), and the average number of detached cells was consistent between the four weeks ( Figure 8 B), where approximately one-third of the initially seeded cells detached every 24 hours. The number of cells harvested from the microcarriers (on which they were previously grown) did increase from day 1, however these cells did not overgrow the microcarrier surface ( Figure 8 D).

[0230] As observed in the static system, both (i) detached cells that detached during the process (yield) and (ii) adherent cells recovered from the microcarriers at day 27 (grown on microcarriers) were able to reattach to the tissue culture plastic after collection and displayed an initial undifferentiated phenotype ( Figure 9 A). Finally, the detached (yield) cells were able to maintain their functionality after several days of cryopreservation ( Figure 9 B).

[0231] Dispase type I

[0232] Different concentrations of Type I Dispase were tested on C2C12 cells grown in serum-free medium. Batch desorption was achieved by supplementing the medium with 0.8 U / mL (equivalent to 0.062 mg / mL) of the Type I Dispase used herein. Starting with 0.8 U / mL (equivalent to 0.062 mg / mL) of Type I Dispase and performing serial dilutions, the inventors found that 0.0008 U / mL (equivalent to 0.00006 mg / mL (60 ng / mL)) enabled them to continuously desorb C2C12 over a period of 5 days. This preliminary experiment suggests that Type I Dispase can be used as a substitute for Type I collagenase (see Figure 11 ).

[0233] Collagenase type VII (pure collagenase)

[0234] The present inventors also tested pure collagenase (type VII collagenase) to see 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 (type I collagenase). The optimal concentration of type VII collagenase was determined to be in the range of 106.35 U / mL to 53.18 U / mL [see Figure 12 ]. Therefore, pure collagenase can also continuously detach cells.

[0235] Example 2

[0236] This example provides support for the cysteine protease ficin and The data for the sequential detachment of adherent cells using collagenase and dispase can be compared.

[0237] The data were generated using C2C12 cells as a cell model grown in serum-free medium (DMEM F12 with 1% insulin-transferrin factor and 1 mM L-ascorbic acid). The data presented below show that ficin and Can:

[0238] i) detached cells in a dose-dependent manner;

[0239] ii) detaching cells in a steady state without changing the cell phenotype; and

[0240] iii) Batch detachment of cells without compromising cell viability.

[0241] The two plant-based cysteine proteases tested were ficin (product FSM200) purchased from Enzybel International SA and (Product PSM100). Ficin and papain ( derived from papain) are cysteine proteases (they have a sulfhydryl group at their active site).

[0242] Ficin

[0243] Ficin (historically known as ficin) is purified from the latex of the fig tree, Ficus glabrata or Ficus anthelmintica, and is part of the cysteine endopeptidase family. This ficin is a food-grade, naturally occurring protease. Optimal working conditions are pH 5-9 and temperatures 40-75°C. The CoA (Certificate of Analysis) from Enzybel indicates an activity unit of 213 bAPA / g for this batch (*). This enzyme has been used in food production and pharmaceutical applications.

[0244]

[0245] Enzyme® is an enzyme preparation (peptidase-papain) derived from papaya; it is food grade and contains endopeptidase. CoA from Enzybel expresses the activity units of this batch as 103 TU / mg (**). It has been used in food and beverages as well as pharmaceutical applications. Source: Papaya (Garica papaya L.)

[0246] *Principles of the BAPA method:

[0247] To analyze the enzymatic (proteolytic) activity of ficin, the synthetic substrate N-α-benzoyl-DL-arginine-p-nitroanilide hydrochloride (DL-BAP(N)A) is used, which is cleaved by the enzyme. The substance "p-nitroaniline" (cleavage fragment) provides an indication of the hydrolysis rate during the reaction. This substance is measured photometrically. One unit of ficin activity corresponds to the amount of enzyme that hydrolyzes 1 μmol substrate equivalent per minute under the assay conditions.

[0248] **The principle of TU method:

[0249] The enzyme solution is added to a buffered solution of casein and digested at 40°C and pH 6.0 for 60 minutes. The reaction is terminated and excess casein is precipitated with trichloroacetic acid. The digest is filtered and the optical density of the clarified filtrate is read at 280 nm. The enzyme activity is then calculated from the observed optical density and the slope of the standard tyrosine curve. The TU unit (TU / mg) is defined as the unit that releases 1 μg of tyrosine per minute when acting on the casein substrate under specific conditions.

[0250] 2.1 Dose-dependent cell detachment

[0251] Similar to collagenase and dispase, ficin and Both enzymes were able to detach cells in a dose-dependent manner. Thus, increasing enzyme concentration resulted in more cells detaching from the surface. Figure 13 Images of cells grown on surfaces after 6 days of incubation with different enzyme concentrations are shown. The ficin used ( Figure 13 A) The concentrations of 0.06 μg / mL, 0.6 μg / mL and 60 μg / mL were used. ( Figure 13 B) The concentrations were 0.07 μg / mL, 0.7 μg / mL, and 70 μg / mL. As the enzyme concentration increased, the number of detached cells increased (numerical data not shown, but can be found in Figure 13), leaving fewer cells growing on the original surface. Thus, there was a dose-dependency between enzyme concentration and the number of detached cells. Both enzymes were confirmed ( Figure 13 ).

[0252] 2.2 Continuous cell detachment

[0253] Similar to collagenase and dispase, ficin and The collected data showed that in serum-free medium, in a static system, C2C12 cells continued to grow and detach within 10 days ( Figure 14 ).

[0254] Figure 14 The images in Figure 2 show (A) 15.36 μg / mL (0.0033 BAPA U / mL) of ficin and (B) 7.68 μg / mL (0.79 TU U / mL) of ficin. A similar confluence was maintained on the proliferation surface (attached) for more than 10 days, while cells continued to detach therefrom (detached).

[0255] Furthermore, immunostaining showed that this procedure did not perturb the phenotype of attached and detached cells. In fact, Pax7 positivity was found to be over 90% in all conditions (data not shown).

[0256] 2.3 Batch cell detachment

[0257] Similar to collagenase and dispase, once added to cell culture medium, ficin and In this experiment, C2C12 cells were detached at 50,000 / cm 2 Cells were seeded at a density of 1 mL in a 24-well plate containing 1 mL of serum-free medium (SFM). The cells were allowed to adhere to the surface overnight. Each enzyme was then prepared in SFM and 1 mL was added to each well. The concentration of ficin ranged from 29.6 mg / mL (6.3 B A P A U / mL) to 1.85 mg / mL (0.39 B A P A U / mL), and The concentration range of the enzymes was 36.625 mg / mL (3772 TU U / mL) to 2.29 mg / mL (236 TU U / mL). Within 30 minutes after addition, the two enzymes tested (ficin 29.6 mg / mL (i.e. 6.3 BAPA U / mL) and 36.625 mg / mL (i.e. 3772 TU U / mL) can effectively detach cells from the surface ( Figure 15 ). Both enzymes were Figure 15 ) complete desorption occurs.

[0258] Cell viability

[0259] Additionally, during the total 4 h incubation with enzymes, ficin and Does not damage cell viability. Collect detached cells, centrifuge and resuspend in serum-free medium (SFM) for cell counting and viability. When desorbed by 1% β-catenin and 1% β-catenin, the viabilities of the desorbed cells were found to be 90.5% and 98.5%, respectively. Figure 16 ).

[0260] in conclusion

[0261] When added to the culture medium at low concentrations, ficin and Able to continuously detach adherent cells in steady state without changing cell viability and phenotype.

Claims

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

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

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

4. The method of claim 1 or 2, wherein the cysteine protease is of the CA family, the cysteine protease is optionally selected from the group consisting of: ficin, papain, bromelain, cathepsin K and calpain, the cysteine protease is further optionally selected from the group consisting of: ficin and papain. The method according to claim 4 , wherein the collagenase is type I collagenase or type VII collagenase. The method of claim 3 , wherein the dispase is a type I dispase.

7. The method according to any one of the preceding claims, wherein step (i) is performed for at least 3 days.

8. The method according to any one of the preceding claims, wherein the cell population is cultured on a support in the cell culture medium, wherein collagenase and / or dispase continuously detaches a portion 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 beads, microcarriers, microfluidic chips, silicon chips, microscope slides, microplate wells, matrices, resins, biochips, multiwell plates, gels, films, and membranes.

11. The method according to 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 portion of the cells from the cell aggregate.

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

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

14. The method of any one of the preceding claims, further comprising regulating the concentration or frequency of the metalloproteinase and / or cysteine protease to regulate 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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