Culture method

Abstract

The present invention relates to a method for reducing aggregation in a cell cluster-based cell culture system, such as a microcarrier-based or spheroid-based cell culture system.

Description

Technical Field

[0001] The present invention relates to a method for reducing aggregation in a cell cluster-based cell culture system, such as a microcarrier-based or spheroid-based cell culture system. Background Art

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

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

[0004] Therefore, in order to sustain such a 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 cell yields have increased significantly, other key production steps are still being optimized. For example, improved cell harvesting methods are needed to make it easier, more efficient, and less invasive to recover expanded cells from the cell culture matrix.

[0006] In order to expand the cell of production for industrial process or the culture system of cell-based therapy, it is generally advantageous to promote the growth of cells in cell clusters (such as microcarriers or spheroids) in bioreactors.Spheroid culture systems overcome the limitations of traditional monolayer cell culture by promoting cell-to-cell and cell-to-matrix interactions, thereby providing similar in vivo physicochemical environment.Microcarriers are particles suspended in cell culture medium, and their effect is to effectively increase the available surface area that cells can adhere to and grow.Microcarriers can generally be spherical particles, and hundreds of cells can grow thereon.By providing such a large surface area for cell proliferation, microbeads provide an environment in which cells can precipitate, so that even in a suspended culture environment, adherent cells such as stem cells or progenitor cells can also grow.

[0007] However, the aggregation or clumping of cell clusters in cell culture (such as spheroids or cell-filled microcarriers) can reduce the cells' access to nutrients and may lead to reduced growth.

[0008] It is an object of the present invention to provide a method for reducing the aggregation or clumping of cell clusters, such as spheroids or cell-filled microcarriers, thereby ameliorating some of the problems associated with the prior art. Summary of the Invention

[0009] In one aspect of the present invention, there is provided a method for reducing aggregation in a cell culture system based on cell clusters, the method comprising the steps of:

[0010] (i) culturing a cell population comprising two or more cell clusters in a cell culture medium for a culture period, wherein the cell culture medium comprises an exogenous protease, wherein the exogenous protease is an exogenous metalloprotease and / or a cysteine protease.

[0011] In one aspect of the present invention, there is provided a method for reducing aggregation in a cell cluster-based cell culture system, the method comprising the steps of:

[0012] (i) culturing a cell population comprising two or more cell clusters in a cell culture medium for a culture period, wherein the cell culture medium comprises a supplemental protease, wherein the protease is a metalloprotease and / or a cysteine protease.

[0013] In another aspect of the present invention, a cell culture vessel is provided, comprising a cell culture medium, two or more cell clusters and an exogenous protease, wherein the exogenous protease is an exogenous metalloprotease and / or a cysteine protease, optionally wherein the two or more cell clusters comprise a cell population in logarithmic growth phase.

[0014] In another aspect of the present invention, there is provided a cell culture vessel comprising a cell culture medium, two or more cell clusters and a supplemental protease, wherein the protease is a metalloprotease and / or a cysteine protease, optionally wherein the two or more cell clusters comprise a population of cells in logarithmic growth phase.

[0015] Suitably, in method of the present invention and / or cell culture container, two or more cell clusters can each comprise a microcarrier. Such cell clusters are also referred to herein as cell-filled or cell-loaded microcarriers. Therefore, suitably, two or more cell clusters can each be a cell-filled microcarrier.

[0016] Suitably, in the methods and / or cell culture vessels of the invention, the two or more cell clusters may each be a spheroid, an organoid or a tissue.

[0017] Suitably, in the methods of the invention, aggregation may be cell-mediated aggregation.

[0018] Suitably, in the methods of the invention, the culturing step may include all or part of the logarithmic phase of growth of the cell population.

[0019] Suitably, in the methods of the invention, the incubation period may be at least 24 hours.

[0020] Suitably, in the methods of the invention, the cell culture system may be a continuous cell culture system or a batch cell culture system, optionally wherein the system comprises a bioreactor.

[0021] Suitably, in the cell culture vessel of the present invention, the vessel may be part of a continuous cell culture system or a batch cell culture system, optionally wherein the system comprises a bioreactor.

[0022] Suitably, in the methods and / or cell culture vessels of the present invention, the metalloprotease may be collagenase and / or dispase.

[0023] Suitably, in the methods and / or cell culture vessels of the invention, the metalloprotease may be a collagenase.

[0024] Suitably, in the methods and / or cell culture containers of the present invention, the collagenase may be 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. It is noteworthy that type I to type VII collagenases are enzyme compositions comprising collagenases of increasing purity, while type VII collagenase is a pure collagenase.

[0025] Suitably, in the methods and / or cell culture vessels of the present invention, the collagenase may be 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.

[0026] Suitably, in the methods and / or cell culture vessels of the invention, the metalloprotease may be a dispase.

[0027] Suitably, in the methods and / or cell culture vessels of the invention, the dispase may be dispase I or dispase II.

[0028] Suitably, in the methods and / or cell culture vessels of the invention, the cysteine protease may be of the CA family.

[0029] 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.

[0030] Suitably, in the methods and / or cell culture vessels of the invention, the two or more cell clusters may each comprise a microcarrier, and the microcarriers may be beads.

[0031] Suitably, in the methods and / or cell culture vessels of the invention, the two or more cell clusters may each comprise a microcarrier, and the microcarriers (such as beads) may comprise a material selected from the group consisting of: plastic, polymer, glass and metal.

[0032] Suitably, in the methods and / or cell culture vessels of the present invention, the cell population may comprise cells selected from the group consisting of: skin, muscle, cervical, breast and prostate cells.

[0033] Suitably, in the methods of the invention, the cell culture medium may be supplemented one or more times with a protease (such as collagenase and / or dispase) during the culturing step.

[0034] Suitably, the method of the invention may comprise culturing the cell population in a cell culture medium lacking metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as ficin and / or papain) before and / or after step (i).

[0035] Suitably, in the methods and / or cell culture vessels of the present invention, metalloproteases (such as collagenase and / or dispase) cysteine proteases (such as ficin and / or papain) may be present in the cell culture medium at a concentration that maintains the cell culture at about 50% to about 100% confluence.

[0036] Suitably, in the methods of the invention, the culturing step may not include a step of washing the cells (e.g., in some instances, it does not include a step of washing the artificial support (e.g., microcarriers) on which the cells are grown, nor a step of washing the cell clusters themselves (e.g., it does not include a step of washing the spheroids, organoids, or tissues, or a step of washing the cell-filled microcarriers).

[0037] Suitably, in the methods of the invention, metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as ficin and / or papain) may be present in the cell culture medium at concentrations that allow for the continuous detachment of a portion of the cells from the cell clusters.

[0038] Suitably, in the methods of the invention, metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as ficin and / or papain) may be present in the cell culture medium at a concentration that allows a ratio of cell detachment rate (e.g. detachment from cell clusters, such as spheroids or microcarriers) to cell growth rate in the range of 0.3:1 to 1:3.

[0039] Suitably, in the methods of the invention, shed cells or a portion of shed cells may be harvested at least once during the culturing step.

[0040] Suitably, in the methods of the invention, shed cells or a portion of shed cells may be harvested more than once during the culturing step.

[0041] Suitably, the methods of the invention may comprise modulating the concentration and / or frequency of administration of a metalloprotease (e.g. collagenase and / or dispase) and / or cysteine protease (e.g. ficin and / or papain) to modulate the rate of cell shedding, wherein increasing the concentration and / or frequency of administration increases the rate of continuous cell shedding and decreasing the concentration and / or frequency of administration decreases the rate of continuous cell shedding.

[0042] Suitably, in the methods of the invention, the cell culture system may comprise a bioreactor.

[0043] Suitably, in the methods and / or cell culture vessels of the present invention, the cell culture system may comprise a population of cell clusters (eg a population of (cell-filled) microcarriers, spheroids, organoids or tissues). BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Embodiments of the present invention are further described below with reference to the accompanying drawings, in which:

[0045] Figure 1 This study demonstrates that collagenase can successfully detach C2C12 cells in significant amounts, similar to TrypLE (using collagenase type I as an example). The graph shows that TrypLE and collagenase exhibit similar numbers of detached cells (A) and survival rates (B), without altering cell adhesion (C), proliferation (D), and differentiation (E) potential (scale bar 200 μm).

[0046] Figure 2 It shows that collagenase can cause cell detachment in a dose-dependent manner without changing the cell phenotype (using type I collagenase as an example). Images show adherent cells (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 number of type I collagenase (in mg / mL) is: 2.635U / mL (0.012mg / mL); 3.500U / mL (0.016mg / mL); and 4.375U / mL (0.020mg / 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 adherent cells, while differentiated cell markers (MHC) are not expressed (scale bar 200μm).

[0047] Figure 3 Schematic diagram showing collagenase supplementation as a strategy for continuous detachment of adherent cells in two systems: on flat and microcarrier surfaces and under two conditions (static and dynamic). Type I collagenase is used as an example.

[0048] Figure 4 Shows continuous shedding in a flat static system. Cell shedding was performed using 1.75 U / mL (equivalent to 0.008 mg / mL) of collagenase (type I), showing similar numbers of adhered (A) and detached (B) cells over 3 weeks. Quantification shows similar numbers of adhered (C) and detached (D) cells over time, indicating steady-state cell proliferation and cell yield (scale bar 200 μm).

[0049] Figure 5 Demonstrating continuous detachment in a flat dynamic system. Example using type I collagenase. (A) Images show that the number of cells adhering to the flat dynamic system is similar over the course of a week. (B) Quantification shows that the number of cells harvested on day 7 is similar to that of cells seeded on day 0. (C and D) Representative images show expression of the undifferentiated cell marker Pax7 in detached (C) and adherent (D) cells (scale bar 200 μm).

[0050] Figure 6 Figure 3. Continuous detachment of cells from microcarrier surfaces when culture medium is supplemented with collagenase (using collagenase type I as an example). Stained nuclei demonstrate that microcarrier aggregation in the control (B) is caused by cell overgrowth, 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 microcarriers at the end of the experiment (scale bars 200 μm and 1000 μm).

[0051] Figure 7 Cell behavior demonstrating adhesion. Cells detached from microcarriers using collagenase (A) can reattach to a flat tissue culture-treated surface in serum plus medium, and cells recovered from microcarriers at the end of the experiment (B). (C) Cells detached using collagenase can also adhere to fresh microcarriers and maintain a continuous process in serum-free medium containing collagenase. Example using collagenase type I (scale bar 200 μm).

[0052] Figure 8 Demonstrating continuous shedding in a dynamic microcarrier system. Quantification of cells shed daily (A) and weekly (B) over 27 days in a dynamic system using collagenase. (C) Representative images of cells stained for nuclear staining on microcarriers and (D) final quantification showing the number of cells seeded and harvested at the end of the experiment. Collagenase type I was used as an example.

[0053] Figure 9Cell behavior showing adhesion. Cells detached from microcarriers using collagenase (detached) can reattach to a flat tissue culture treated surface in serum plus medium, as well as cells recovered from microcarriers at the end of the experiment (adherent). (C) Cells detached by collagenase can be cryopreserved and, once thawed (thaw-freeze), they can reattach and grow on tissue culture treated cell culture plastic. Collagenase type I (scale bar 200 μm) is used as an example.

[0054] Figure 10 Demonstrating that collagenase can successfully induce the shedding of large numbers of fish skin fibroblasts. After 20 hours of incubation with collagenase, the cells detach (A), and after collection (B), they are able to reattach (5 hours after inoculation, C) and proliferate (48 hours after inoculation, D). This example uses type I collagenase at a concentration of 1505 U / mL (equivalent to 7 mg / mL, scale bar 500 μm).

[0055] Figure 11 Dispase was shown to successfully induce continuous detachment of C2C12 cells. While the confluence of growing cells was maintained over a 3-day period (adherence, A), the cells detached at a comparable rate over time (detachment, B). Further experiments also demonstrated the same effect over 21 days (data not shown). Dispase I was used at a concentration of 0.0008 U / mL (equivalent to 0.00006 mg / mL or 60 ng / mL, scale bar 200 μm) as an example.

[0056] Figure 12 Collagenase was shown to successfully induce continuous detachment of C2C12 cells. The confluence of growing cells (adherent) was maintained over a 3-day period, while cells detached at a comparable rate over time (detached). Collagenase type VII was used as an example. (A) Collagenase VII was used at a concentration of 106.35 units / mL (equivalent to 0.07 mg / mL); (B) Collagenase VII was used at a concentration of 53.18 units / mL (equivalent to 0.035 mg / mL, scale bar 200 μm).

[0057] Figure 13 Showing ficin and The images show the detachment of C2C12 cells in serum-free medium with different concentrations of ficin (A) and (B) Growth on the surface after 6 days of incubation (scale bar 250 μm).

[0058] Figure 14 Shown with ficin (A) or (B) Continuous cell shedding. Images taken on day 0 and day 10 show the initial confluence (after cell seeding) and the confluence after continuous enzyme exposure, respectively. The image on day 10 shows the confluence of cells on the growth surface (adherent) and shed during the last 3 days of the experiment (shed, scale bar 250 μm).

[0059] Figure 15 Shown with ficin or Once added to the culture medium, C2C12 cells can be treated with ficin (A) and (B) Extensive shedding. Cells had already shed after 30 minutes, and were completely shed after 150 minutes (scale bar 250 μm).

[0060] Figure 16 showed that after exposure to ficin or The cell survival rate was then increased by ficin or A large number of detached C2C12 cells maintained high cell viability after exposure to high enzyme concentrations for 4 h. DETAILED DESCRIPTION

[0061] definition

[0062] As used herein, "about" will be understood by one 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 one of ordinary skill given the context in which it is used, "about" means up to ±0.1% of the specified value.

[0063] As used herein, the terms "comprise," "comprises," and "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components of one aspect or embodiment 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 groups thereof.

[0064] The phrase "consisting essentially of" means that the scope of an aspect or embodiment should be interpreted as including the specified materials or steps described in that aspect or embodiment, including any materials or steps that do not materially affect the invention as defined in that aspect or embodiment. Therefore, the term "consisting essentially of" used in the claims of the present invention should not be interpreted as equivalent to "comprising."

[0065] The term "cell cluster" refers to a group (e.g., a sphere or layer, etc.) of cells. It refers to a plurality of adjacent or interconnected cells. A cell cluster can be formed, for example, by at least 10 adjacent cells (where each cell is in direct contact (in other words, touching) with at least one other cell in the cluster). For example, the cluster can include 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 example, the adjacent cells are interconnected.

[0066] As used herein, " adjacency " refers to the cell that is connected to each other in the mode of forming cell ball, layer etc..When being placed in the solution such as cell culture medium, adjacency cell keeps cluster (for example ball, layer etc.).Adjacency cell can directly contact, and for example wherein they adhere to each other or touch in the mode of forming cell cluster.Alternatively, adjacency cell can be connected indirectly in the mode of forming cell cluster, for example, by the existence of matrix, support or support (for example extracellular matrix), wherein matrix, support or support connect adjacent cells into cluster.Term " cell cluster " also can be referred to as cell aggregate in this article.The example of cell cluster comprises cell layer (for example on microcarrier), spheroid, organoid, tissue or its any combination.

[0067] Those skilled in the art will understand that cells can form cell clusters with or without artificial supports. In one example, cells can form cell clusters without artificial supports (such as cell clusters can be spheroids, organoids or tissues, wherein cell growth and / or mutual adhesion, in other words, cell self-aggregation forms cell clusters). In another example, due to growth on identical artificial supports (such as cell growth on microcarriers, wherein cell growth and / or adhesion on the support (and optionally growth and / or mutual adhesion), cells can be adjacent cells (i.e. cell clusters). Term " cell clusters " herein includes all of these examples, therefore includes cells / cell layers adhered to the artificial supports, such as microcarriers (such as cell-filled microcarriers), and spheroids, organoids or tissues (such as microtissues).

[0068] Spheroids are spherical cell units that are typically cultured as free-floating aggregates with low complexity in mirroring tumor tissue. Spheroids are carefully designed cell aggregates that can be used as engineered cancer models. They have the ability to promote 3D construction of tumor growth to amplify tumor cell progression and help reveal new anti-cancer treatments. Tumor spheroids can be derived from cancer cell lines and may have the potential to be used as a medium for 2D culture models and in vivo tissue xenografts. Tumor spheroids known to be produced by permanent cancer cell lines are avascular. They can induce micrometastasis formation and may show cancer-like microenvironmental characteristics (such as cell-cell, cell-ECM exchange, local nutrient transport, gas and growth factors). As used herein, the term "spheroid" also includes tumor spheroids, also referred to herein as tumor organoids. Generally, organoids can be referred to as 3D grown cells to form structural units that are similar to organ parts in structure and function.

[0069] Organoids are 3D-derived stem cell models derived from embryonic or adult stem cells. Organoids can exhibit the potential for self-regulation, grow phenotypic characteristics of the organ they are derived from, and physiologically model their environment through genomic alterations. In addition, they can mimic in vivo 3D structures and have multi-lineage differentiation capabilities into different tissue types (e.g., airway organoids engineered from lung-derived stem cells to study lung physiology, patterning, and lung morphogenesis).

[0070] The cell clusters described herein are typically cultured in suspension (they are suspended in a cell culture medium). In other words, they do not adhere to the cell culture vessel in which they are cultured. Thus, the cell culture systems described herein can be referred to as cell cluster-based cell culture systems, in which cell clusters (e.g., cells on microcarriers, spheroids, organoids, tissues, etc.) are in suspension during the culture period.

[0071] The cell clusters described herein typically include adherent cells. As used herein, the term "adherent cells" refers to a homogeneous or heterogeneous population of cells that are anchorage-dependent, i.e., they require attachment to an artificial support or other cells (e.g., in the form of a cell cluster) to grow in vitro. Suitably, in the context of the present disclosure, the artificial support may be a microcarrier.

[0072] The methods of the present invention enable users to reduce the aggregation of cell clusters (e.g., cell-filled microcarriers, spheroids, etc.) during cell culture. In this context, "aggregation" refers to the formation of one or more groups of cell clusters (aggregates), wherein one group (aggregate) may contain two or more cell clusters that would otherwise exist alone in the cell culture. For example, "aggregation" can refer to the formation of one or more groups of cell-filled microcarriers (aggregates), wherein one group (aggregate) may contain two or more cell-filled microcarriers that would otherwise exist alone in the cell culture. In another example, "aggregation" can refer to the formation of one or more groups of spheroids (aggregates), wherein one group (aggregate) may contain two or more spheroids that would otherwise exist alone in the cell culture. In another example, "aggregation" can refer to the formation of one or more groups of organoids (or tissues) (aggregates), wherein one group (aggregate) may contain two or more organoids (or tissues) that would otherwise exist alone in the cell culture. Aggregation in this context typically occurs due to interactions between cell clusters. Aggregation is also referred to herein as "aggregation" or "aggregation between cell clusters."

[0073] In the context of the present disclosure, the term "cell-mediated aggregation" refers to the aggregation of cell clusters due to cell-cell interactions (e.g., between cells of two or more different cell clusters to induce aggregation), cell-ECM interactions (e.g., between the ECM of one cell cluster and cells of a different cell cluster) and / or cell-microcarrier interactions.

[0074] In some embodiments, cell-cell interaction can occur between the cells cultivated (or adhered) on different artificial supports. In some embodiments, cell-cell interaction can occur between the cells cultivated (or adhered) on different microcarriers, thereby causing microcarrier aggregation. When the cells on different microcarriers adhere to each other or touch (i.e. interconnected) in the mode of forming cell-filled microcarrier aggregates, cell-cell interaction can occur. As used herein, " interconnection" refers to the cell that directly contacts each other and is for example physically connected by intercellular connection (for example, by one or more cells connecting (also referred to as intercellular bridge (a plurality of))). Cell connection is made up of multiprotein complex, provides contact between adjacent cells or between cell and extracellular matrix. Cell connection is especially abundant in epithelial tissue. Cell connection enables communication between adjacent cells.

[0075] In another example, cell-cell interactions may occur between cells of different cell clusters that do not contain an artificial support, resulting in cell cluster aggregation. For example, cell-cell interactions may occur when cells on different spheroids adhere to or touch each other (i.e., interconnect) in a manner that forms a spheroid aggregate. In another example, cell-cell interactions may occur when cells on different organoids (or tissues) adhere to or touch each other (i.e., interconnect) in a manner that forms an organoid (or tissue) aggregate.

[0076] Suitably, cell-microcarrier interactions may occur between cells adhered to (or cultured on) a first microcarrier interacting with a second microcarrier, thereby promoting or maintaining aggregate formation.

[0077] In some examples, the cells in a cell cluster are all of the same type. For example, they might all be brain cells, muscle cells, or heart cells. In other examples, the cells in a cell cluster are all from the same lineage, such as all hematopoietic progenitor cells. In some examples, the cells are stem cells, such as neural stem cells or embryonic stem cells.

[0078] The cell cluster-based cell culture system as described herein can be used in continuous bioprocessing systems.

[0079] Thus, in one example, a cell cluster includes homogeneous or heterogeneous cell types."Cell growth"refers to the division or proliferation of seeded cells.

[0080] " artificial support " mentioned herein refers to the surface of any suitable supporting biomaterial, for example cell, cell mass, cell culture, tissue or fluid or other biomaterial or composition, for example those described herein. Support can be called surface, can be applicable to carrying process or reaction, for example cell or organism, cell mass, cell culture or tissue growth and development. In the context of the present disclosure, artificial support can be microcarrier. " microcarrier " is a kind of particle, is suitable for supporting cell adhesion and / or growth thereon. The microcarrier with the cell that adheres (or cultivates) thereon is referred to as cell-filled microcarrier in this article.

[0081] A "gel" is a semisolid, jelly-like substance that does not flow in its solid state. Gels comprise a 3D cross-linked network that provides the gel with its semisolid structure. Gels can be hydrogels, which contain a network of insoluble but hydrophilic polymer chains. Gels can be defined based on their viscoelastic or rheological properties.

[0082] 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.

[0083] As used herein, "cell viability" refers to the ability of a cell to remain metabolically active to promote growth and function.

[0084] As used herein, the term "culturing" refers to maintaining cells in cell culture under conditions suitable for growth for a period of time. This period of time may be referred to herein as the "culture period."

[0085] As used herein, in the context of cell clusters comprising artificial supports, "confluence" refers to the percentage of supports (e.g., microcarriers) covered by cells (e.g., adherent cells). A 50% confluence means that half of the supports (e.g., microcarriers) are covered by cells (e.g., adherent cells). A 100% confluence or complete confluence means that all supports (e.g., microcarriers) are covered by cells (e.g., adherent cells). Excessive confluence means that there is no space available for new cells on the support (e.g., microcarriers).

[0086] In the context of cell clusters that do not include artificial supports (such as spheroids, organoids or tissues), cell density or cell cluster size is more important. Methods for determining cell density and size are well known in the art and are described elsewhere herein.

[0087] As used herein, the term "cell culture medium" refers to a nutrient solution used to culture living cells to allow the cells to proliferate.

[0088] As used herein, the term "exogenous protease" refers to a protease added to a cell culture medium (i.e., the cell culture medium has been supplemented with a protease). It refers to a protease that is not expressed by the cells present in the cell culture medium (these proteases are considered endogenous proteases herein). Therefore, it is a protease from an external source (it is not naturally produced in this amount by the cells in the cell culture medium). For the avoidance of doubt, the cells in the cell culture medium may also be able to produce these proteases, however, the proteases referred to herein as exogenous proteases are proteases that are added to the cell culture medium from an external source. Exogenous proteases may also be referred to herein as cell culture medium supplements or supplemental proteases. Suitably, in the context of the present disclosure, an exogenous protease is an exogenous metalloprotease and / or cysteine protease.

[0089] As used herein, the term "metalloprotease" refers to a protease that has one or more metal ions at the binding / active site. Examples of metalloproteases include collagenase and / or dispase.

[0090] The term "cysteine protease" is intended to describe proteases that have a highly reactive thiol group of a cysteine residue in the catalytic site of the enzyme. Cysteine proteases are known in the art and may be referred to herein as "thiol proteases" or "sulfhydryl proteases". Many superfamilies of cysteine proteases are known in the art. Suitably, in the context of the present disclosure, the cysteine protease may be of the CA family (sometimes also referred to as papain-like proteases). Papain-like proteases have a common catalytic binary active site characterized by a cysteine amino acid residue acting as a nucleophile. Suitably, the cysteine protease of the CA family may be selected from 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 / ).

[0091] Suitably, papain may be formulated as A powdered preparation of purified papain, standardized with maltodextrin. Papain is an enzyme extracted from the unripe green fruit of the papaya tree.

[0092] Ficin, sometimes also called ficain, is a proteolytic enzyme typically extracted from the latex sap of the stems, leaves, and unripe fruit of the American wild fig tree, the plain fig (Ficus inspida).

[0093] 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 appropriately defined herein. Contacting can include bringing the two or more items into close physical relationship and / or into contact with each other. Contacting is sometimes referred to herein as "exposing," e.g., exposing a cell to a cysteine protease should be understood as bringing the cell into contact with the cysteine protease.

[0094] "Seeding" cells means applying an initial cell population to a cell culture medium or support."Seeded cells" are cells that are initially applied to a support.

[0095] The term "shedding" or "shedding" with respect to cells on an artificial support such as a microcarrier refers to the separation of the cell from the support such as a microcarrier such that it is no longer anchored or adhered to the support such as a microcarrier.

[0096] The term "shedding" or "shedded" with respect to a cell cluster (e.g., a spheroid, organoid, or tissue) that does not comprise an artificial support refers to the separation of a cell from the cell cluster (e.g., a spheroid, organoid, or tissue) such that it is no longer anchored or adhered to the cell cluster (e.g., a spheroid, organoid, or tissue).

[0097] As used herein, "frequency of administration" refers to the number of times an agent, particularly a protease (such as collagenase and / or dispase) is added to the cell culture medium before and / or during the cell culture period.

[0098] The "logarithmic growth phase" is the logarithmic or exponential phase of cell growth, during which cells actively proliferate and cell density increases. The logarithmic phase typically follows a lag phase and precedes a stationary phase, during which growth slows or stops.

[0099] The term "suspension" or "suspension culture" is a type of cell culture in which cells are grown in small clusters in an agitated growth medium or on artificial supports such as microcarriers.

[0100] The term "cell cluster-based cell culture system" refers to a cell culture system in which cells are cultured in suspended cell clusters, rather than, for example, on the surface of a cell culture flask. The term "microcarrier-based cell culture system" refers to a cell culture system in which cells are cultured using microcarriers on which they are cultured, rather than, for example, on the surface of a cell culture flask. The term "spheroid-based cell culture system" refers to a cell culture system in which cells are cultured in spheroids, rather than, for example, on the surface of a cell culture flask. The term "organoid (or tissue)-based cell culture system" refers to a cell culture system in which cells are cultured in organoids (or tissues), rather than, for example, on the surface of a cell culture flask.

[0101] As used herein, in the context of reducing aggregation in a cell culture system based on cell clusters, the term "reduction" refers to reducing the proportion, number or size of aggregates formed in the cell culture compared to a suitable control. The reduction can be, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more compared to the control. Suitably, the reduction can be about 100% compared to the control. In the context of the present disclosure, a control can be the level of aggregation in a method comprising the following steps: (i) culturing a cell population comprising two or more cell clusters in a cell culture medium for a culture time, wherein the cell culture medium does not contain an exogenous protease.

[0102] These methods may also be referred to herein as "reducing" aggregation in cell cluster-based cell culture systems. DETAILED DESCRIPTION

[0104] The present invention provides a method for reducing aggregation in a cell cluster-based cell culture system, comprising the following steps: (i) culturing a cell population comprising two or more cell clusters in a cell culture medium for a culture time, wherein the cell culture medium comprises an exogenous protease, wherein the exogenous protease is an exogenous metalloprotease and / or a cysteine protease.

[0105] Suitably, the aggregation of two or more cell clusters in cell culture may be cell-mediated aggregation.

[0106] The present invention is based on a surprising discovery that, by reducing the excessive growth of cells and / or preventing the bridging between cell clusters in the cell culture medium, the aggregation in the cell culture system based on cell clusters can be reduced. The present invention is based on providing metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) in the cell culture medium appropriately during the growth phase, which destroy the cell-cell interaction between different cell clusters in the cell culture and / or cause a portion of cells to fall off from the cell cluster to prevent excessive growth. Therefore, the present invention provides a method for reducing the aggregation of microcarriers, spheroids, organoids or tissues filled with cells in suspension culture during cell culture. The method of the present invention also provides a method for controlling the convergence of cells on artificial supports (such as microcarriers). As a result, cells can be prevented from reaching a high degree of convergence and / or excessive growth. Therefore, the present invention provides a novel method for reducing or actually preventing aggregation (aggregation) during cell culture. Surprisingly, the inventors have found that other types of proteases, such as serine proteases (such as trypsin), do not have the same effectiveness in the context of the present invention due to causing a large amount of fall-off. Without wishing to be bound by this hypothesis, the inventors believe that this is due to the different mechanisms of action of metalloproteinases and cysteine proteases compared to serine proteases. Specifically, serine proteases can transmit intracellular signals, while metalloproteinases and cysteine proteases act on the extracellular matrix rather than with / on the cells and do not directly trigger any intracellular response.

[0107] The presence of metalloproteinases and / or cysteine proteases in the cell culture medium reduces the aggregation of cell clusters (aggregation of microcarriers, spheroids, organoids, tissues, as appropriate, of cells). In the case of the presence of exogenous metalloproteinases and / or cysteine proteases in the cell culture medium, the ratio of the cell clusters that gather together can suitably be 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the total number of cell clusters in the cell culture medium. In fact, the presence of aggregates in the cell culture medium may be substantially 0%, which means that protease can prevent the aggregation of cell clusters basically. Therefore, the cell clusters of the desired ratio will remain suspended in the cell culture medium and will not adhere to other cell clusters present in the culture medium. Suitably, the presence of metalloproteinases and / or cysteine proteases in the culture medium also helps to reduce or prevent the combination or aggregation of cell clusters on the surface of a cell culture system (such as a bioreactor). Suitably, the invention provides a cell culture in a bioreactor, wherein the cell clusters remain as a homogenous (suspension) colony in the cell culture medium.

[0108] The methods of the present invention have the advantage of increasing the growth capacity of cell cultures by reducing or preventing aggregation, which can limit cells' access to nutrients and / or gases and lead to reduced or restricted growth. The methods of the present invention also improve cell harvesting from microcarriers by avoiding excessive growth, cell clumping, reducing shearing and / or loss of viable cells due to aggregation.

[0109] The methods of the invention may comprise one or more steps prior to step i). One method may comprise, for example, obtaining a cell population from a suitable source.

[0110] The present invention is particularly 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, cornea, endometrium, esophagus, gastrointestinal tract, immune system (such as T lymphocytes, B lymphocytes, leukocytes, macrophages and dendritic cells), liver, lungs, lymphatic vessels, muscles (such as myocardium), nerves, ovaries, pancreas (such as islet cells), pituitary gland, prostate, kidney, saliva, skin, tendons, testicles and thyroid gland. In some embodiments, cells are mammalian cells (for example, 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 may be selected from myocytes, cardiomyocytes, epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, osteoblasts, chondrocytes, adipocytes, and mesenchymal stem cells. By way of example only, immortalized cells may be selected from HeLa cells, HEK 293 cells, 3T3 cells, A549 cells, Vero cells, CHO cells, OK cells, C2C12 cells, and PTK2 cells. The cells may be disease cells or disease model cells, such as cancer cells or cells in a hyperproliferative state. In one example, the cell population includes cells selected from skin, muscle, cervical, breast, and prostate cells.

[0111] Suitable cells are described elsewhere herein, and those skilled in the art will understand how to obtain such cell populations.For example, the method may comprise obtaining a suitable tissue, and isolating cells therefrom.

[0112] Cells can be cultured as adherent cell populations on artificial supports such as microcarriers.

[0113] Suitably, the cell population on the microcarriers can be at least or no more than 50%, 60%, 70%, 80%, 90% or 100% confluence, meaning that at least or no more than 50%, 60%, 70%, 80%, 90% or 100% of the microcarrier surface area is occupied by cells. When nearly all of the surface area of the microcarriers is occupied by cells, the cell layer can be referred to as confluent. In one example, the presence of metalloproteinases and / or cysteine proteases in the cell culture medium prevents the cell layer on the microcarriers from converging or over-converging, or maintains confluence at a desired level.

[0114] Suitably, during the cell culture period, the cell layer is maintained at 80%-100% confluence. Suitably, the confluence on the microcarriers does not exceed 50%, 60%, 70%, 80%, 90%, 95% or 100%. The confluence on any two or more microcarriers in the cell culture system of the present invention may be the same or different. The confluence on any two or more microcarriers in the cell culture system of the present invention may be, for example, in the range of 5% to 95% confluence, for example 80% to 100% confluence.

[0115] During the defined cell culture period, cells can be continuously detached, meaning that according to the method of the present invention, when metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) present in the cell culture medium cause the cells to continuously detach from the cell clusters in the cell culture medium, the stationary growth phase is not reached.

[0116] The methods of the present invention have numerous advantages. For example, the methods can increase the yield of cells in cell culture by maintaining the cells in the logarithmic growth phase for a longer period of time. Furthermore, the methods offer the advantage of reducing the amount of resources required to produce the desired yield, thereby reducing the resource or carbon footprint.

[0117] When microcarriers are used in the context of the present invention, the cell layer in contact with the microcarriers can be a single layer of cells (meaning that the layer is one cell deep), or more than one cell layer (two or more cells deep). The cells can include one cell type, or two or more different cell types.

[0118] 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. Prior to step i), if the cells are cultured or maintained in a cell culture medium, this may be done in the absence of exogenous proteases.

[0119] The present invention includes cultivating a cell group comprising a plurality of cell clusters. Each cell cluster can be a cell-filled microcarrier. Mention that microcarriers can include a single microcarrier or a microcarrier group. A group can include two or more microcarriers. The microcarriers in a single group (single cell culture) can all be the same or different, for example in size, shape, material, porosity, density and / or surface modification. Suitable microcarriers or microcarrier combinations for the present invention can be selected by technicians based on factors including cell type to be cultivated and desired culture results. If necessary, microcarriers can be modified or adjusted to support or improve cell adhesion to it.

[0120] The microcarriers can be placed in a suitable growth chamber or container.

[0121] Prior to use, the microcarriers and / or suitable growth chamber or container may typically be sterilized, for example, by gamma irradiation, autoclaving, alcohol washing, or treatment with ethylene oxide (ETO) gas.

[0122] The microcarrier can be a particle, such as a bead. The microcarrier can be spherical or ovoid. The microcarrier can include a planar surface on which the cell population can be cultured.

[0123] Microcarriers can be rigid or elastic. Microcarriers can be porous or impermeable. Non-natural porous materials can be made porous by methods available to the skilled artisan, such as sintering, etching, leaching, photolithography, or laser micromachining. An example of a porous support can be a gel or a mesh. Microcarriers can be edible.

[0124] In one example, the microcarrier comprises or consists of DEAE dextran, glass, polystyrene, plastic, PEG, acrylamide, or natural polymers such as collagen, chitin, and its derivatives, and cellulose, hyaluronic acid, alginate, PLGA, or gelatin, or any combination thereof.

[0125] Should be understood that all or part of the microcarrier can be made of the material that promotes cell adhesion or use it to process.Microcarrier can be made of the material that supports cell adhesion, or can carry out modification to support cell adhesion on its whole or part of surface.The material that promotes cell adhesion can be selected from 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 loofah (vegetable sponge).Suitably, cellulose can be cellulose acetate.Extracellular matrix components can be one or more in fibronectin, vitronectin, cartilage adhesion protein or laminin.Suitably, bonding material is electrostatic.Suitably, bonding material can be collagen or gelatin. Suitably, the adhesive material may comprise a peptide amphiphile (PA), as described by Miotto et al. in Developing a Continuous Bioprocessing Approach to Stromal Manufacture, ACS Applied Materials & Interfaces 2017 9(47), 41131-41142. Adhesive cells may adhere to the surface of the microcarrier via an anchoring substrate such as an integrin or other cell receptor. The microcarrier may comprise a non-adhesive portion.

[0126] Microcarriers of the same material may differ in their porosity, specific gravity, optical properties, presence of animal components, and surface chemistry or modification. https: / / en.wikipedia.org / wiki / Specific_gravitySurface chemistry can include the presence of extracellular matrix proteins, functional groups, recombinant proteins, peptides, and positively or negatively charged molecules added to the microcarrier surface by conjugation, copolymerization, plasma treatment, or grafting. https: / / en.wikipedia.org / wiki / Functional_group https: / / en.wikipedia.org / wiki / Peptides https: / / en.wikipedia.org / wiki / Conjugation_(pharmacokinetics) https: / / en.wikipedia.org / wiki / Copolymer

[0127] The microcarriers may have a diameter in the range of 50 μm to 1 mm, preferably about 100 μm in diameter. It will be appreciated that the size and composition of the microcarriers may be determined by the type of cells to be cultured on the microcarriers. The selection of suitable microcarriers is well within the capabilities of those skilled in the art.

[0128] The present invention includes culturing a cell population comprising a plurality of cell clusters. Each cell cluster may be a spheroid. Reference to a spheroid may include a single spheroid or a cluster of spheroids. A cluster may include two or more spheroids. The spheroids within a single cluster (single cell culture) may be identical or different, for example, in size, shape, and / or density. The spheroids may be placed in a suitable growth chamber or container.

[0129] In another example, each cell cluster can be an organoid (or tissue). References to organoids (or tissues) can include single organoids (or tissues) or groups of organoids (or tissues). A group can include two or more organoids (or tissues). The organoids (or tissues) in a single group (single cell culture) may be the same or different, for example in size, shape and / or density. The organoids (or tissues) can be placed in a suitable growth chamber or container.

[0130] The methods of the present invention can be performed in a growth chamber. Those skilled in the art will appreciate that "growth chamber" refers to any suitable chamber or container suitable for growing cells. It will be understood that the growth chamber can include microcarriers and cell culture medium. Suitably, the growth chamber can be a bioreactor.

[0131] Bioreactor is any suitable chamber for accommodating the cell clusters described herein, suitably in suspended form. Therefore, bioreactor can have any suitable surface type and any suitable geometry. Therefore, suitable bioreactor can include but is not limited to hollow fiber, stirred tank, gas lift, bubble column or fluidized bed bioreactor, packed bed bioreactor or flexible bag. Bioreactor can be liquid phase, gas phase or mixed bioreactor. Bioreactor can be disposable or multiple use. It can be a benchtop bioreactor or an industrial bioreactor. Bioreactor can be horizontal or vertical and can have an agitator or be affected by waves or rocking motion. Therefore, it is obvious that any suitable growth chamber (such as bioreactor) can also be used.

[0132] Before step i), a suitable cell culture medium can be selected. The cell culture medium can be a complete preparation (i.e., a cell culture medium that does not require nutritional supplements for the cultured cells), or it can be an incomplete preparation (i.e., a cell culture medium that requires nutritional supplements), or it can be a culture medium that can supplement an incomplete preparation, or in the case of a complete preparation, it can improve the culture or culture results. Those skilled in the art will know various cell culture media, and they will also understand that the type of cell to be cultured can determine the type of culture medium to be used. Suitably, a cell culture medium that does not significantly affect the activity of selected metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) is selected.

[0133] By way of example only and not limitation, the cell culture medium may be selected from Dulbecco's Modified Eagle's Medium (DMEM), Ham's F-12 (F-12), Leibovitz's L-15 medium, RPMI-1640, Mesencult TM Basal medium, minimal essential medium (MEM), basal medium Eagle (BME), Ham's F-10, alpha minimal essential medium (αMEM), Glasgow minimal essential medium and Iscove's modified Dulbecco's medium (IMDM) or any combination thereof. In the context of the present disclosure, other commercially available culture media (e.g., from Thermo Fisher Scientific of Waltham, Massachusetts) or other culture media known in the art can be used equivalently. Similarly, by way of example only, the culture medium can be selected from 293SFM, CD-CHO culture medium, VP SFM, BGJb culture medium, Brinster's BMOC-3 culture medium, cell culture freezing medium, CMRL culture medium, EHAA culture medium, eRDF culture medium, Fischer culture medium, Gamborg's B-5 culture medium, GLUTAMAX TM Supplemented medium, 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), NCTC-109 medium, 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, Complete methylcellulose medium, HepatoZYME-SFM, Neurobasal TMCulture medium, Neurobasal medium-A, Hibernate TM A medium, hibernation 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.

[0134] Suitably, the cell culture medium may be serum-free. Suitably, the cell culture medium may be glucose-free.

[0135] Alternatively, the cell culture medium may contain serum. Suitable types and amounts of serum are known, for example 1% FBS may be used.

[0136] Suitably, the cell culture medium may contain one or more additional agents as required. These may be selected from, but not limited to, antibiotics, buffers, growth factors, hormones, nutritional supplements, indicators, and essential metals and minerals.

[0137] Prior to step i) in the next step, the cells may be seeded on the microcarriers. Suitably, the cells are seeded onto the microcarriers after incubation of the microcarriers with the cell culture medium. Methods of cell seeding are known and available to those skilled in the art and may be adjusted depending on the cell type and support. Two or more microcarriers may be seeded simultaneously. The initial cell seeding density must be effective while allowing for optimal cell proliferation within the microcarriers. The number of cells to be seeded also depends on the porosity of the scaffold material and any liquid absorption capacity. For porous microcarriers, 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 In addition, the porosity of the scaffold and the internal organization of the support fibers help the cells to be retained in and on the support. The method can include more than one sequential seeding step on the support.

[0138] Metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be provided in the cell culture medium before the cell culture medium is applied to the cell culture. Therefore, the cell culture medium comprising metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to the support or growth chamber itself. Alternatively, metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be provided to the cell culture medium separately. Suitably, metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) are added to the cell culture before the incubation period begins. Therefore, 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 lag phase of cell growth.

[0139] 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 near 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%.

[0140] Additional metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to cell cultures during incubation. Supplemental metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to cell cultures once or more than once during incubation. For example, supplemental metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to cell cultures 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 times or more can be added to cell cultures during cell culture. Supplemental metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can be added to cell cultures at regular or irregular intervals. In some embodiments, the metalloproteinases (e.g., collagenase and / or dispase) and / or cysteine proteases (e.g., papain and / or ficin) are administered at regular intervals. For example, regular 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 additional metalloproteinases (e.g., collagenase and / or dispase) and / or cysteine proteases (e.g., papain and / or ficin) can be referred to as "pulses." If supplementary metalloproteinases (e.g., collagenase and / or dispase) and / or cysteine proteases (e.g., papain and / or ficin) are added at irregular intervals, these may depend on factors such as the growth rate of the cell, the shedding rate, the degree of confluence on the microcarriers, and / or the concentration of metalloproteinases (e.g., collagenase and / or dispase) and / or cysteine proteases (e.g., papain and / or ficin) in the cell culture medium. When the shedding rate is lower than the growth rate, e.g., lower than 10%, 20%, 30%, 40% or 50% or more, supplemental metalloproteases (e.g., collagenase and / or dispase) and / or cysteine proteases (e.g., papain and / or ficin) may be provided to the cell culture.

[0141] 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 this embodiment, a certain amount of existing cell culture medium can be removed to maintain the desired concentration of proteases (such as collagenase and / or dispase). Suitably, removing or adding cell culture medium does not equate to washing the cells and does not change the cell growth phase in the logarithmic phase. During the cell culture phase, the cell culture medium is not completely removed and wash medium is not added to the cell culture.

[0142] The cells are cultured under appropriate cell culture conditions for at least a period of time. Those skilled in the art will appreciate appropriate cell culture conditions for different cell types. Typical growth conditions are 37°C, 95% relative humidity, and 5% CO availability. The pH can be maintained between 7 and 7.4.

[0143] Cell cultures can be maintained under static or dynamic conditions. In a dynamic system, motion is imposed on the cell culture (e.g., by being placed on a rocker) to allow movement of the cell culture medium. Agitation has the effect of maintaining cells and / or cell clusters suspended in the cell culture medium. In a static system, there is no mechanism to provide movement of the cell culture medium. In such a system, cell clusters can form a static bed.

[0144] 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 of time between the lag phase and the stationary phase, wherein the cells in the culture are actively proliferating. Suitably, the culture period comprises all or part of the logarithmic phase, suitably comprises 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. The growth phase of cell culture can be determined using any suitable method, such as visual or analytical methods (such as cell counting, DAPI staining etc.).

[0145] Suitably, the 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, the cell culture can continue for 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, the cell culture can be about 1 day to about 9 days, such as about 2 days to about 8 days, or about 3 days to about 7 days.

[0146] Suitably, the cells may 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 may be cultured for at least about 40 days.

[0147] 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.

[0148] In the method for the present invention, suitably, cell (for example on microcarrier) will not reach confluence during the cell culture phase, or suitably will not become excessive confluence. Suitably, method for the present invention enables cell to keep the confluence of about 50% to about 100%, more suitably 60% to 95%, more suitably 80% to 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%. Suitably, during the cell culture phase, confluence remains on less than 100%. In another example, during the cell culture phase, confluence can remain on about 100% or less than about 100%.

[0149] Confluency can be maintained at the levels described herein during the cell culture period by the use of metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) in the cell culture medium, which allows for continuous detachment of cells (e.g., from a support or from other adherent cells, such as from cell aggregates) as the cells proliferate and the population grows.

[0150] In the method of the present invention, metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can cause a portion of cells to continuously fall off from the cell cluster. In this context, "causing a portion of cells to continuously fall off" refers to continuously removing a portion of cells from a cell cluster (e.g., removing a portion of a cell cluster on a microcarrier, or removing a portion of a cell cluster in a spheroid, organoid, or tissue). Therefore, shedding occurs gradually over time rather than as a discrete (large amount of shedding) event.

[0151] Those skilled in the art will understand that continuous shedding occurs over a period of time (in the incubation period, typically more than 24 hours). Therefore, in the incubation period, metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) cause a portion of cells to continuously fall off from the cell cluster in the cell mass. Typically, the method of the present invention includes causing adherent cells to fall off from the cluster in the cell mass (using exogenous metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin)) while the cell mass is undergoing growth phase (typically when the cell mass is in the logarithmic phase). Therefore, continuous shedding can be used to maintain the cell mass at a confluence or density that enables the cell mass to remain in the logarithmic growth phase (or at least avoid the stationary phase and lag phase of cell growth) in the incubation period. This may be particularly useful in the context of a continuous bioprocessing system (such as a bioreactor). Thus, during step (i), the methods of the present invention typically maintain the adherent cell population at a confluence of 40% to 95% (e.g., when adhered to a support). Optimal confluence is discussed elsewhere herein and will be readily determined by one skilled in the art.

[0152] Thus, during the culture period, metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can cause a portion of cells (e.g., at least 5% but not more than 60%) to continuously detach from the cell clusters in the cell population.

[0153] Thus, during the culture period, metalloproteases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) can cause a portion of cells (e.g., at least 0.5% but not more than 7% per hour) to continuously detach from the cell clusters in the cell population.

[0154] In the methods of the present invention, continuous shedding can maintain the cell confluence or density of cells in the cell clusters 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).

[0155] In the methods of the present invention, continuous shedding can maintain the cell confluence or density of cells in the cell clusters during the culture period (in other words, in some examples, the cell confluence or density can increase or decrease by no more than 40% on average per week during the culture period).

[0156] It will be clear to those skilled in the art that "continuous shedding" does not include bulk shedding (a term well known in the art). In the context of exogenous metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin), bulk shedding can refer to shedding of at least 60% of the cells in a cell cluster within 30 minutes at 37°C. Therefore, step (i) of the method of the present invention does not include bulk shedding of cells in a cell cluster.

[0157] Suitably, the shedding rate exceeds the proliferation rate during the culture period.

[0158] Suitably, the concentration of the metalloproteinases (such as collagenase and / or dispase) and / or cysteine proteases (such as papain and / or ficin) provided in the cell culture medium allows the shedding rate of cells (from cell clusters, for example from microcarriers, or from other adherent cells, for example from spheroids, organoids, tissues) to be in the range of 0.3:1 to 1:3 relative to the cell growth rate. In other words, the shedding rate may be one-third or more of the proliferation rate. The proliferation rate may be up to 3 times of the shedding rate. The shedding rate and proliferation rate can be determined by, for example, imaging, bioreactor sensors / probes, analytical methods, all of which are well known in the art.

[0159] Suitable, during the cell culture period, can gather in the crops at least one exfoliated cell or a part of exfoliated cell. Suitable, during the cell culture period, can gather in the crops more than one exfoliated cell or a part of exfoliated cell. Harvest exfoliated cells can be carried out once 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, remove the suitable method of exfoliated cells and comprise using dead end or cross flow / tangential fluid flow filtration, centrifuge, acoustic separation or based on the system of microfluidic. Alternatively, do not gather in the crops exfoliated cells, and can remain in the cell culture, until the cell culture period ends.

[0160] Before or after step i) of the inventive method, cells can be passaged one or more times. Therefore, the method can include removing cells from cell culture, and placing the cells on, for example, new microcarriers. Suitably, the cells are cell populations that come off by the method of the present invention.

[0161] The method of the present invention may also include monitoring or determining the survival rate of cells in the cell culture. Cell survival rate can be measured by measuring cell proliferation or metabolic activity. Other methods include flow cytometry and immunohistochemistry. Cell survival rate can be measured after the culture period in step i).

[0162] Suitably, in the methods of the invention, the metalloprotease (such as collagenase and / or dispase) and / or cysteine protease (such as papain and / or ficin) does not substantially adversely affect the viability of the cell population (e.g., over a culture period of at least 24 hours, such as at least 48 hours, such as at least 72 hours). In this context, a 10% decrease in cell viability over the culture period, compared to the cell viability observed in the absence of the metalloprotease (such as collagenase and / or dispase) and / or cysteine protease (such as papain and / or ficin), may be considered a substantial adverse effect.

[0163] Method of the present invention can also include monitoring or determining the confluence (for example, on support) of cell.Cell confluence can use chemical dye (for example, thymidine, Alamar blue, XTT or other dyestuffs available in this area), qualitative visual measurement state or use image processing method to measure, and this image processing method comprises for example Olympus CKX53 culture microscope, CKX-CCSW confluence inspection software and air fraction output.As described in following example, Cytation 1, IoLight, Jiusion USB digital microscope and ImageJ can also be used to image and analyze cell.Show that the method cell is more than expected confluence, then extra protease (such as collagenase and / or dispase) can be added in cell culture medium to increase the shedding rate of cell in cell cluster.Can consider the total concentration of protease (such as collagenase and / or dispase) to remain in the optimal limit value of cell type.

[0164] Cell confluence can also be referred to as the density of adherent cells (e.g., cell density on a carrier or in a cell cluster). Cell density can be determined using, for example, a microscope, acoustic resonance density determination, laser induced fluorescence, fluorescence microscopy, capacitance impedance, turbidity, biomass dielectric constant probes, Raman probes, and cell counters, such as CCD imaging using Trypan blue. Cell density can also be used to determine the shedding rate of cells in a cell cluster, as described elsewhere herein.

[0165] The methods of the invention may comprise monitoring or determining the growth phase of cells in culture. This may be performed using visual means, for example examining cell shape.

[0166] 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.

[0167] The method of the present invention may comprise repeating step i) two or more times.

[0168] Suitably, the incubation period in step i) does not include a step of washing the cells. A washing step may be included after step i). Depending on the choice of cells (e.g., the choice of cell clusters), washing may be performed using any suitable method. Washing may include aspirating the cell culture medium and placing the cells in a physiological buffer.

[0169] The methods of the invention may comprise counting cells.

[0170] Suitably, substantially all cells of the cell population do not differentiate during the cell culture period.Thus, substantially all cells of the cell population do not show signs of differentiation, such as markers of differentiation or changes in cell shape or morphology.

[0171] The methods of the present invention may further comprise culturing the cells under conditions suitable for differentiation.

[0172] The methods of the present invention may also include cryopreservation of the harvested cells.

[0173] Any suitable source of exogenous metalloproteinases (e.g., collagenase and / or dispase) and / or cysteine proteases (e.g., papain and / or ficin) can be used in the present invention. Suitably, the metalloproteinases (e.g., collagenase and / or dispase) and / or cysteine proteases (e.g., papain and / or ficin) are compatible with the cell culture medium. Suitably, the metalloproteinases (e.g., collagenase and / or dispase) and / or cysteine proteases (e.g., papain and / or ficin) are capable of causing cell shedding without substantially affecting cell growth or survival.

[0174] 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.

[0175] In one example, type I collagenase may therefore contain other enzymes such as tyrosine kinase, clostripain and trypsin. Suitably, type I collagenase may have greater than 125 U / mg of collagenase; greater than 200 U / mg of tyrosine kinase; less than 4 U / mg of clostripain and less than 0.5 U / mg of trypsin activity.

[0176] If the metalloprotease is a collagenase, it may be obtained from Clostridium histolyticum, which is also used in human medicine. However, it may also be isolated from other Clostridium bacteria or tissues (e.g. Merck Index No. 2477).

[0177] Suitable collagenases may be Gibco TMType I collagenase (Fisher Scientific product code 11500536), isolated from Clostridium histolyticum, contains average levels of collagenase, tyrosinase, clostripain, and trypsin activities. This is the type I collagenase used in the following examples.

[0178] Many FDA-approved collagenases are known in the art and available. For example, these include santalyl salicylate (Smith and Nephew); cellulite (Qwo, Endo International) and clostridium histolyticum collagenase (Endo International).

[0179] Suitably, a single metalloprotease (i.e., collagenase or dispase) or cysteine protease (i.e., papain or ficin) can be used for cell culture during a single cell culture period. Alternatively, a combination of two or more proteases that are metalloproteases or cysteine proteases can be provided in the cell culture during a single cell culture period. By way of example only, one metalloprotease (i.e., collagenase or dispase) and one cysteine protease (i.e., papain or ficin) can be used for cell culture during a single cell culture period, or a combination of two or more metalloproteases (e.g., collagenase and dispase) or cysteine proteases (i.e., papain and ficin) can be used for cell culture during a single cell culture period. Combinations of metalloproteases and cysteine proteases can also be used for cell culture during a single cell culture period.

[0180] It will be clear to those skilled in the art that 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, as long as the protease reduces the aggregation of cell clusters 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 their common knowledge. The exact amount to be used may depend on many factors, such as the type of cell, cell culture medium, whether static or dynamic conditions are used, whether cells need to be detached, etc. Some non-limiting examples are provided herein. For example, studies have shown that under serum-free conditions, static culture requires a higher concentration of collagenase compared to dynamic culture in a flat system. When using microcarriers, even higher concentrations are required. When serum is present in the cell culture medium, it is also observed that the concentration increases further (using 9 to 20 U / mL of type I collagenase to achieve steady static-steady-state continuous detachment; compared to using 5 to 10 U / mL to achieve steady dynamic-steady-state continuous detachment; a large amount of detachment requires 4200 U / mL (20 mg / mL)-data not shown). Therefore, those skilled in the art can determine the appropriate concentration. Suitably, when using collagenase, 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.

[0181] 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.

[0182] Suitably, type I collagenase may be provided in the cell culture at 2 U / mL or less.

[0183] 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, be not more than, be lower than or be higher than any value of above-mentioned scope.In each case therein, 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 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, where the collagenase is type I collagenase, it may be provided in the cell culture at a concentration of 5 U / mL to 10 U / mL, or at a concentration of 0.008 U / mL to 5 U / mL.

[0184] Suitably, when the collagenase is type VII collagenase, it can be provided in cell culture at 500 U / mL or less. Suitably, type VII collagenase is provided in cell culture at 300 U / mL or less. Suitably, type VII collagenase is provided in cell culture at 200 U / mL or less. Suitably, type VII collagenase is provided in cell culture at 150 U / mL or less. Suitably, type VII collagenase can be provided in cell culture at 120 U / mL or less. Suitably, type VII collagenase can be provided in cell culture at 110 U / mL or less.

[0185] In each case therein, the lower limit of scope can be 50U / mL.For example, when collagenase is collagenase type VII, it can be provided in cell culture with a concentration of 50U / mL to 300U / mL.For example, when collagenase is collagenase type VII, it can be provided in cell culture with 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 with a concentration of 50U / mL to 120U / mL.For example, when collagenase is collagenase type VII, it can be provided in cell culture with a concentration of 50U / mL to 110U / mL.

[0186] Suitably, where the enzyme is dispase, it may be provided in the cell culture medium at 13 U / mL or less. Suitably, dispase is present in the cell culture medium at 6.5 U / mL or less.

[0187] Suitably, dispase is present in the cell culture medium with 3U / mL or lower. Suitably, dispase is present in the cell culture medium with 2U / mL or lower or 1U / mL or lower. Suitably, dispase is present in the cell culture medium with 0.8U / mL or lower or 0.5U / mL or lower. In each case therein, the lower limit of the scope can be 0.0005U / mL, for example 0.0008U / mL. For example, dispase can be provided in the cell culture with a concentration of 0.0008U / mL to 3U / mL. For example, dispase can be provided in the cell culture with a concentration of 0.0008U / mL to 2U / mL or with a concentration of 0.0008U / mL to 1U / mL. For example, dispase can be provided in the cell culture with a concentration of 0.0008U / mL to 0.8U / mL. For example, dispase can be provided in the cell culture with a concentration of 0.0008U / mL to 0.5U / mL.

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

[0189] When expressed as enzyme concentration, exogenous metalloproteases (such as collagenase and / or dispase) may be provided in the cell culture medium at 1 mg / mL or less, or suitably at 0.5 mg / mL or less. Suitably, the exogenous protease (such as collagenase and / or dispase) 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. Suitably, the metalloprotease (such as 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 / mL, or any range formed by any of the upper or lower limits of the aforementioned ranges, or any integer therebetween, or at least, no more than, less than, or greater than any value in the aforementioned ranges.

[0190] Suitably, the exogenous cysteine protease (such as 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 the 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 / mL, or any range formed by any of the upper or lower limits of the above ranges, or any integer therebetween, or at least, no more than, less than, or greater than any value in the above ranges. In another example, ficin can be provided in the 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, for example, about 0.0005 to about 0.01, about 0.001 to about 0.009 or for example 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. Suitably, papain 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 0.75 mg / mL, 0.001 mg / mL to 0.5 mg / mL, 0.005 mg / mL to 0.25 mg / mL or any range formed by any one in the upper or lower limit of the above range or any integer therebetween or at least, not more than, less than or higher than any value in the above range. In another example, papain can be provided in the cell culture medium at a concentration of about 0.0001 mg / mL to 0.07 mg / mL, such as about 0.001 mg / mL to about 0.025 mg / mL.

[0193] Suitably, papain can be provided in the cell culture medium at a concentration of about 0.001 to about 10 TU U / mL, such as about 0.05 to about 5, about 0.1 to about 2, or such as about 0.5 to about 1 TU U / mL. Suitably, papain can 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 cultured meat industry. Suitably, the methods of the present invention can include processing steps related to the use of the cells produced in cell culture.

[0195] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including but not limited to," and are not intended to (and do not) exclude other parts, additives, components, integers, or steps. Unless the context requires otherwise, throughout the description and claims of this specification, the singular encompasses the plural. 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.

[0196] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except combinations where at least some of those features and / or steps are mutually exclusive. The invention is not limited to the details of any foregoing embodiments. The invention extends to any novel feature or any combination of novel features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel method or process or any novel combination of steps of any method or process so disclosed.

[0197] The reader's attention is directed to all papers and documents which are filed concurrently with or before this specification in connection with this application and which are open to public inspection with this specification, the contents of all of which are incorporated herein by reference.

[0198] Example

[0199] Example 1

[0200] 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).

[0201] 1.1 Collagenase can successfully induce the detachment of C2C12 cells without changing their adhesion, proliferation and differentiation potential.

[0202] Collagenase has been previously used for mass detachment of certain types of adherent cells as an alternative to more common methods such as trypsin and TrypLE. To achieve mass detachment of cells in a short period of time, 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; this is equivalent to 2.2 mg / mL). Collagenase supplementation was effective in detachment efficiency ( Figure 1A) and the survival rate of cells collected after detachment ( Figure 1 B) showed comparable results to TrypLE. In addition, similar to control cells, cells detached by collagenase could reattach, proliferate, and differentiate ( Figure 1 CE).

[0203] 1.2. Collagenase can cause cell detachment in a dose-dependent manner.

[0204] It has been demonstrated that increasing amounts of collagenase lead to the detachment of increasing numbers of C2C12 cells (Table 1).

[0205] Table 1: Quantification shows that the use of type I collagenase (units / cell) increases the number of detached cells in a dose-dependent manner.

[0206]

[0207] This suggests that collagenase can surprisingly be used to control the rate of cell shedding. Furthermore, the detached cells were able to reattach and there was no differentiation during this process ( Figure 2 ).

[0208] 1.3. Low concentration of collagenase can cause C2C12 cells to detach continuously.

[0209] It has been demonstrated that administration of collagenase at lower concentrations can achieve continuous cell detachment without affecting cell properties. This was studied in four different systems (see A to D below).

[0210] A. Continuous cell shedding in a flat system under static conditions.

[0211] It has been demonstrated that supplementation with 1.75 U / mL (equivalent to 0.0081 mg / mL of type I collagenase used herein) can enable continuous growth and detachment of C2C21 cells in static culture in serum-free medium for 21 days ( Figure 4 ). Exfoliated cells were collected every 24 hours and counted on days 1, 7, 9, 14, and 21. The confluence on the growth surface remained similar over time ( Figure 4 A) did not grow 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).

[0212] B. Continuous shedding in a flat system under dynamic conditions.

[0213] It has been demonstrated that supplementation with 0.009 U / mL of type I collagenase (equivalent to 0.00004 mg / mL (43.27 ng / mL) of type I collagenase used herein) can enable continuous growth and detachment of C2C12 cells in dynamic culture in serum-free medium for 7 days ( Figure 5 ). Debonded 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 adherent cells harvested was similar to the number of cells seeded ( Figure 5 B). In addition, at the end of 7 days of continuous flow culture, both adherent and detached cells showed an initial undifferentiated phenotype ( Figure 5 C and Figure 5 D).

[0214] C. Continuous shedding in a microcarrier system under static conditions .

[0215] It has been demonstrated that supplementation with 4.5 U / mL of type I collagenase enables continuous growth and detachment of C2C12 cells in static culture in serum-free medium for 27 days.

[0216] 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). Shedding cells were collected every 24 hours, and the average number of shed cells remained consistent between the first and second weeks ( Figure 6 D), approximately one-third of the initial seeded cells were shed every 24 hours. The number of cells harvested from the microcarriers (previously grown on the microcarriers) did increase from day 1, but the cells did not overgrow on the microcarrier surface, resulting in aggregation ( Figure 6 E).

[0217] Furthermore, (i) exfoliated (derived) cells that were shed during the process and (ii) adherent cells (grown on microcarriers) recovered from the microcarriers at day 27 were able to re-adhere to tissue culture plastic after collection and displayed an initial undifferentiated phenotype ( Figure 7 A and Figure 7 B). In addition, the detached (generated) cells were able to re-adhere to fresh microcarriers in the presence of collagenase and undergo the same process again ( Figure 7 C).

[0218] D. Continuous shedding of microcarrier systems under dynamic conditions.

[0219] It has been demonstrated that supplementation with 4.5 U / mL of type I collagenase enables continuous growth and detachment of C2C12 cells in dynamic culture in serum-free medium for 27 days. Cell detachment was consistently observed during the collagenase-supplemented culture. Figure 8). The shed cells were collected every 24 hours ( Figure 8 A), the average number of cells shed remained consistent over four weeks ( Figure 8 B), approximately one-third of the initial seeded cells were shed every 24 hours. The number of cells harvested from the microcarriers (previously grown on the microcarriers) did increase from day 1, but these cells did not overgrow on the microcarrier surface ( Figure 8 D).

[0220] As observed in the static system, (i) detached (generated) cells that were shed during the process and (ii) adherent cells (grown on microcarriers) recovered from the microcarriers at day 27 were able to re-adhere to the tissue culture plastic after collection and displayed an initial undifferentiated phenotype ( Figure 9 A and Figure 9 B). Finally, the shed (generated) cells were able to maintain their function after being cryopreserved for several days ( Figure 9 C).

[0221] Dispase I

[0222] Different concentrations of Dispase I were tested on C2C12 cells grown in serum-free medium. Extensive shedding was achieved by supplementing the culture medium with 0.8 U / mL (equivalent to 0.062 mg / mL) of the Dispase I used herein. Starting with serial dilutions of Dispase I at 0.8 U / mL (equivalent to 0.062 mg / mL), the inventors found that 0.0008 U / mL (equivalent to 0.00006 mg / mL (60 ng / mL)) allowed them to continuously shed C2C12 over a 5-day period. This preliminary experiment suggests that Dispase I can be used as a substitute for type I collagenase (see Figure 11 ).

[0223] Collagenase VII (pure collagenase)

[0224] The inventors also tested pure collagenase (collagenase VII) to see if it could also be used to achieve continuous cell detachment. Various concentrations were tested and compared to the crude reagent (collagenase type I), continuous cell detachment was achieved using higher enzyme units. The optimal concentration of collagenase VII was determined to be between 106.35 U / mL and 53.18 U / mL [see Figure 12 ]. Therefore, pure collagenase was also able to cause continuous cell detachment.

[0225] Example 2

[0226] The data provided in this example support the hypothesis that the cysteine proteases ficin and Can be used similarly to collagenase and dispase to continuously detach adherent cells and reduce microcarrier aggregation.

[0227] 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). Can:

[0228] i) caused cell shedding in a dose-dependent manner;

[0229] ii) shedding cells in a steady state without altering cell phenotype; and

[0230] iii) to achieve high cell shedding without compromising cell viability.

[0231] The two plant-based cysteine proteases tested were ficin (product FSM200) and (Product PSM100), purchased from Enzybel International SA. Ficin and papain ( derived from papain) are cysteine proteases (they have a sulfhydryl group in their active site).

[0232] Ficin

[0233] Ficin (historically known as Ficain) is purified from the latex of the fig tree (Ficus glabrata) or (Ficus anthelmintica). It is a member 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. Enzybel's CoA (Certificate of Analysis) indicates an activity of 213 bAPA / g (*). This enzyme has been used in food production and pharmaceutical applications.

[0234]

[0235] This is an enzyme preparation derived from papaya (peptidase-papain); it is food grade and contains endopeptidase. The CoA of Enzybel indicates that the activity units of this batch are 103 TU / mg (**). It has been used in food and beverages as well as pharmaceutical applications. Origin: Papaya (Garica papaya L.)

[0236] *Principle of BAPA method:

[0237] To analyze the enzymatic (proteolytic) activity of ficin, a synthetic substrate, N-α-benzoyl-DL-arginine p-nitroanilide hydrochloride (DL-BAP(N)A), is used, which is cleaved by the enzyme. The substrate "p-nitroanilide" (the cleaved fragment) indicates the rate of hydrolysis during the reaction. This substrate is measured photometrically. One unit of ficin activity corresponds to the amount of enzyme that hydrolyzes 1 μmol of substrate per minute under the assay conditions.

[0238] **The principle of TU method:

[0239] The enzyme solution is added to a casein buffer solution, and the casein is digested at 40°C and pH 6.0 for 60 minutes. The reaction is stopped and excess casein is precipitated with trichloroacetic acid. The digest is filtered and the optical density of the clarified filtrate is read at 280nm. The enzyme activity is then calculated based on the observed optical density and the slope of the standard tyrosine curve. The TU unit (TU / mg) is defined as the unit that releases 1μg of tyrosine per minute when acting on a casein substrate under specific conditions.

[0240] 2.1 Dose-dependent cell shedding

[0241] Similar to collagenase and dispase, ficin and Both were able to detach cells in a dose-dependent manner, so that as the concentration of the enzyme increased, the number of cells detached from the surface increased. Figure 13 Images of cells grown on a surface after 6 days of incubation at different enzyme concentrations are shown. The concentrations of ficin used ( Figure 13 A) was 0.06 μg / mL, 0.6 μg / mL and 60 μg / mL. The concentration ( Figure 13 B) 0.07 μg / mL, 0.7 μg / mL and 70 μg / mL. As the enzyme concentration increased, the number of cells detached increased (digital data not shown, but can be seen in Figure 13 ), which resulted in a decrease in the number of cells growing on the original surface. Thus, there was a dose-dependent relationship between enzyme concentration and the number of cells detached. This was confirmed for both enzymes ( Figure 13 ).

[0242] 2.2 Continuous cell shedding

[0243] Similar to collagenase and dispase, ficin and Both were able to shed cells in a steady state. The collected data showed that in a static system in serum-free medium, C2C12 cells continued to grow and shed over a 10-day period ( Figure 14 ).

[0244] Figure 14The 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. All were able to maintain similar confluence on the proliferation surface (adherent) for over 10 days while cells continued to shed from the proliferation surface (shedding).

[0245] Furthermore, immunostaining showed that this procedure did not perturb the phenotype of adherent and detached cells. In fact, Pax7 positivity was greater than 90% in all conditions (data not shown).

[0246] 2.3 Massive cell shedding

[0247] Similar to collagenase and dispase, ficin and Once added to the cell culture medium, both can cause a large number of cells to detach. In this experiment, C2C12 cells were cultured at 50,000 / cm 2 The 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 diluted to 29.6 mg / mL (6.3 B A P A U / mL, Figure 15 1 mL of ficin was added to each well at concentrations ranging from A) to 1.85 mg / mL (0.39 BAPA U / mL) and 1 mL of tricinase was added to each well at 36.625 mg / mL (3772 TU U / mL, Figure 15 B) Add 1 mL of Both enzymes at the tested concentrations effectively detached cells from the surface 30 minutes after their addition ( Figure 15 Both enzymes were completely removed after 150 minutes ( Figure 15 ).

[0248] Cell viability

[0249] In addition, adding ficin and Cell viability was not compromised during the total 4-hour incubation with the enzyme. Detachable cells were collected, centrifuged, and resuspended in serum-free medium (SFM) for cell counting and viability. and ficin, the survival rates of the detached cells were found to be 90.5% and 98.5%, respectively. Figure 16 ).

[0250] in conclusion

[0251] When added to the culture medium at low concentrations, ficin and Enables continuous detachment of adherent cells in a steady-state manner without altering cell viability and phenotype.

Claims

1. A method for reducing aggregation in a cell culture system based on cell clusters, the method comprising the following steps: (i) culturing a cell population comprising two or more cell clusters in a cell culture medium for a culture period, wherein the cell culture medium comprises an exogenous protease, wherein the exogenous protease is an exogenous metalloprotease and / or a cysteine protease.

2. The method of claim 1, wherein the two or more cell clusters each comprise a microcarrier.

3. The method of claim 1, wherein each of the two or more cell clusters is a spheroid, an organoid, or a tissue.

4. The method according to any one of the preceding claims, wherein the aggregation is cell-mediated aggregation.

5. The method according to any one of the preceding claims, wherein the culture period of (i) comprises all or part of the logarithmic growth phase of the cell population.

6. The method according to any one of the preceding claims, wherein the incubation period is at least 24 hours.

7. The method according to any one of the preceding claims, wherein the cell culture system is a continuous cell culture system or a batch cell culture system, optionally wherein the system comprises a bioreactor.

8. A cell culture container comprising a cell culture medium, two or more cell clusters, and an exogenous protease, wherein the two or more cell clusters comprise a cell population in a logarithmic growth phase, and wherein the exogenous protease is an exogenous metalloprotease and / or a cysteine protease.

9. The cell culture container of claim 8, wherein the container is part of a continuous cell culture system or a batch cell culture system, optionally wherein the system comprises a bioreactor.

10. The method according to any one of claims 1 to 7 or the cell culture container according to claim 8 or 9, wherein the metalloprotease is collagenase and / or dispase.

11. The method or the cell culture container according to claim 10, wherein the collagenase is type I collagenase or type VII collagenase.

12. The method or cell culture container of claim 10, wherein the dispase is dispase I.

13. The method according to any one of claims 1 to 7 or the cell culture container according to claim 8 or 9, wherein the cysteine protease belongs to the CA family.

14. The method or cell culture container according to claim 13, wherein the cysteine protease of the CA family is selected from the group consisting of ficin, papain, bromelain, cathepsin K and calpain.

15. The method or cell culture container of any preceding claim, wherein the two or more cell clusters each comprise a microcarrier, and the microcarriers are beads.

16. The method or cell culture container of any preceding claim, wherein the two or more cell clusters each comprise a microcarrier, and wherein the microcarrier comprises a material selected from the group consisting of: plastic, polymer, glass, and metal.

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

18. The method according to any one of claims 1 to 7 or 10 to 17, wherein the cell culture is supplemented with the protease one or more times during the culturing step.

19. The method according to any one of claims 1 to 7 or 10 to 18, wherein the method comprises the following steps: Before and / or after step (i), the cell population is cultured in a cell culture medium lacking the protease.

20. The method of any one of claims 1 to 7 or 10 to 19, wherein the protease is present in the cell culture medium at a concentration that maintains the cell culture at about 50% to about 100% confluence.

21. The method of any one of claims 1 to 7 or 10 to 20, wherein the culturing step does not comprise a step of washing the cells.

22. The method of any one of claims 1 to 7 or 10 to 21, wherein the protease is present in the cell culture medium at a concentration that allows for continuous shedding of a portion of the cells in the cell clusters.

23. The method of claim 22, wherein the protease is present in the cell culture medium at a concentration that allows a ratio of cell shedding rate to cell growth rate in the range of 0.3:1 to 1:

3.

24. The method according to any one of claims 22 to 23, wherein the exfoliated cells or a portion of the exfoliated cells are harvested at least once during the culturing step.

25. The method of any one of claims 22 to 24, wherein the method comprises adjusting the protease concentration and / or administration frequency to modulate the cell shedding rate, wherein increasing the concentration and / or the administration frequency increases the continuous cell shedding rate, and decreasing the concentration and / or the administration frequency decreases the continuous cell shedding rate.

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