JP6943449B2 - Proliferation and passage of pluripotent stem cells using agitated tank bioreactor - Google Patents

Description

The present disclosure generally relates to the proliferation and passage of cells and / or cell aggregates using agitated tank bioreactors.

Pluripotent stem cell banking (eg, induced pluripotent stem cells), commercial production of cells (eg, GE CytivaTM myocardial cells), and large-scale pluripotency for application in cell proliferation for clinical trials The need for stem cell culture is emerging. Advances in feeder-free pluripotent stem cell cultures have enabled large-scale cell proliferation on flasks, microcarriers (150-250 microns in diameter) or macrocarriers in bioreactors (about 6 mm in diameter). The use of suspension cultures involves inefficient seeding and release of cells from carriers, physical separation of microcarriers and cells during harvesting, and formation of cell carrier aggregation that can lead to changes in cell phenotype. Avoid some of the challenges that arise when culturing pluripotent cells on traditional microcarriers. Perfusion is typically used for suspension culture in a bioreactor.

However, one challenge in perfusion / suspension culture is how to retain cells in the bioreactor. The prior art provides several basic separation techniques: 1) filtration, 2) gravity sedimentation, and 3) centrifugation. Filtration methods require several means to prevent the filter from clogging over the required weeks of operation. The problem with gravitational sedimentation is the change in sedimentation properties of different cells, the difficulty of scaling up to industrial systems, and the difficulty of maintaining sterility. Similarly, centrifugation is routinely used in open cell cultures, but has only been found in fully closed cell cultures due to sterility concerns.

Techniques are needed in the art to reduce human intervention and cross-contamination during the process of culturing cells, including pluripotent stem cells and / or differentiated human cells.

European Patent Application Publication No. 2966163

Improved methods for culturing cells, including pluripotent stem cells and / or differentiated human cells, are described herein.

Provided herein are methods for the growth of cell aggregates in a closed system.
Steps to provide a cell culture vessel and
Steps to form aggregates in the container,
The step of automatically perfusing cell aggregates in the container,
The step of gravity sedimentation of cell aggregates during perfusion,
Steps to collect and subculture aggregates in a closed system,
including.

Also provided herein is a closed system for use in growing cell aggregates using the methods described herein.

These and other features, aspects, and advantages of the present invention are better understood when the following detailed description is read with reference to the accompanying drawings in which similar letters represent similar parts throughout the drawings. Will be.

FIG. 5 shows a piping assembly for gravity sedimentation and medium replacement in a stirring tank bioreactor in which used medium is vertically removed from the bioreactor. FIG. 5 shows a piping assembly for gravity sedimentation and medium replacement in a stirring tank bioreactor with fresh medium added vertically to the bioreactor. It is a figure which shows the piping assembly for gravity sedimentation and medium exchange in a stirring tank bioreactor which used medium is removed from a bioreactor in an inclined direction. FIG. 5 shows a piping assembly for gravity sedimentation and medium replacement in a stirring tank bioreactor in which fresh medium is added to the bioreactor in an inclined direction. It is a figure which shows the 10mm slicer grid structure having a thickness of 100μm-300μm. It is a figure which shows the square grid slicer which the spacing between walls is 100 μm, and the thickness of a wall is 30 μm. It is a figure which shows the hexagonal grid slicer which the spacing between walls is 100 μm, and the thickness of a wall is 30 μm. It is a figure which shows the method for the closed system treatment of agglomerates through a slicer. Circulation loops driven by pumps and in-line conical bags suspend and distribute aggregates. The pipe leading to the slicer is connected to the main circulation loop, and a part of the cell aggregate is supplied to the slicer via a second pump operating at a low speed. It is a figure which shows the image of the morphology of the sliced aggregate of CT2 human embryonic stem cell. FIG. 5 shows a comparison of proliferation rates after enzymatic or mechanical passage by a slicer on CT2 human embryonic stem cell aggregates seeded at 4x10 ^ 5 cells / mL. FIG. 5 shows a comparison of growth rates after enzymatic or mechanical passage by a slicer on CT2 human embryonic stem cell aggregates seeded at 1.5x10 ^ 6 cells / mL. (1) CT-2 passaged with Accutase® + ROCK inhibitor, (2) CT-2 passaged with square grid + ROCK inhibitor, (3) CT-2 passaged with hexagonal grid + ROCK inhibitor- 2, (4) CT-2 passaged with a square grid + ROCK inhibitor, (5) CT-2 passaged with a hexagonal grid + ROCK inhibitor. It is a figure which shows the morphology of CT2 human embryonic stem cell aggregate in a stirring tank reactor.

The singular form ("A" or "an") means one or more, at least one, herein. When the plural is used herein, it generally includes the singular.

As used herein, "perfusion" refers to the process of surviving cultured cells by continuously feeding the cells in fresh medium and removing the used medium while culturing the cells.

"Aggregate" refers to cell binding in which the binding is caused by cell-cell interaction rather than adhesion to the substrate. In aggregates, two or more cells bind to each other by biological attachment. It can be mediated by surface proteins such as extracellular matrix proteins. In one embodiment, cells can proliferate first on a substrate, with some cells binding (adhering) to the substrate, while further proliferation is binding (adhering) between further grown cells to the substrate. Form cell-cell junctions (aggregation) independent of. In another embodiment, cells spontaneously bind in suspension to form cell-cell adhesion independent of surface adhesion. The cellular feeder layer is also considered as a substrate. Therefore, cell attachment to the feeder layer is also a form of adherent culture (not aggregates), as the cells adhere to the cells in the feeder layer rather than to each other.

"Proliferation" means the proliferation of cells with or without differentiation and can include no passage, one passage or two or more passages and / or continuous passages. In one embodiment, proliferation refers to the proliferation of undifferentiated cells and comprises one passage or two or more passages and / or continuous passages.

"Stem cell" means a cell that is capable of undergoing self-renewal (ie, progeny with the same potency) and producing progeny cells with more limited potency. In the context of the present disclosure, stem cells are more differentiated than undifferentiated, for example, by nuclear transfer, by fusion with more primitive stem cells, by the introduction of specific transcription factors, or by culture under certain conditions. Also includes cells. "Pluripotent stem cells" can potentially produce cells or tissues that the body needs to repair itself. Pluripotent stem cells can also self-replicate and constantly make more replicas. Pluripotent stem cells include induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

"Culture vessels" include disposable and non-disposable plastic products, bags and / or vessels and / or bioreactors. The term includes single-use plastic products, bags and / or containers and / or bioreactors and multiple-use plastic products, bags and / or containers and / or bioreactors.

"Closed system" means a culture vessel and accessories that are pre-sterilized while closed and / or sealed to maintain integrity and / or sterility. Containers and parts are utilized without violating the integrity of the system, allowing fluid transfer and / or outflow while maintaining sterilization, and connectable to other closed systems without loss of integrity. Closed bioreactors and / or vessels refer to systems in which cells, cell culture media, chemicals and reagents are aseptically added, removed and / or manipulated without violating system integrity (eg,). By opening the cap of the tube or lifting the lid from the cell culture plate or dish). Single-use or multi-use bags and / or containers and / or bioreactors within the closed system are added on or in the closed system, eg, at the site of the vessel or bioreactor, by sterile tube welding.

The "subject" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, humans, livestock, sports animals, and pets. Subjects in need of treatment according to the methods of the invention include those who suffer from loss of function as a result of physical or disease-related injuries.

The term "therapeutically effective amount" refers to an amount determined to produce any therapeutic response in a mammal. For example, effective amounts of therapeutic cells or cell-related substances may prolong patient viability. Alternatively, the treatment is prophylactic and can prevent and / or suppress overt clinical symptoms. Therapeutically effective treatments within the meaning of the terms used herein include treatments that improve the subject's quality of life, even if they do not improve the outcome of the disease by themselves. Such therapeutically effective amounts will be confirmed by those of skill in the art through routine application to subject populations such as clinical and preclinical studies. Thus, "treating" means delivering such an amount.

"Treatment," "treating," or "treatment" are widely used with respect to the present invention, and such terms are, among other things, those that impede deficiency, dysfunction, disease or treatment and / or result from treatment. Includes improving, inhibiting, or curing other harmful processes, including.

Large-scale pluripotency for pluripotent stem cell banking (eg, induced pluripotent stem cells), commercial production of cells (eg, GE CytivaTM cardiomyocytes), and / or cell proliferation for clinical trials. Stem cell culture is required. Pluripotent stem cells are induced pluripotent cells (iPS cells), embryo-derived "true" embryonic stem cells (ES cells), embryonic stem cells produced by somatic cell nucleus transplantation (ntES cells), or unfertilized eggs. It may be a derived embryonic stem cell (pluripotent embryonic stem cell or pES cell). Proliferation of large cell feeder-free embryonic stem cells in flasks is labor-intensive, bans space, and isolated populations may exhibit phenotypic drift. Therefore, there are field attempts to develop alternative approaches for large-scale pluripotent stem cell cultures, such as CellSTACK® (Corning), Cell Factory (Nunc®) and Bio. Reactor (microcarrier or suspension culture).

There are many types of bioreactors used for cell culture, including, but not limited to, agitated tank reactors, spinner flasks, orbital shakers, locking motions, and paddle wheels. Those skilled in the art will recognize that there are alternative approaches for cell culture in bioreactors.

Suspended aggregate culture of pluripotent stem cells in an impeller agitated tank bioreactor system has been demonstrated to eliminate the need for any substrate or carrier in bioreactor culture (Chen, VC et. Al, Stem Cell). Res. 2012 May; 8 (3): 388-402). In the Chen method for subculturing cells from suspension culture, aggregates were collected by centrifugation. In contrast, the methods provided herein are closed system proliferation (seed, perfusion, passage and harvesting) of pluripotent stem cells and / or pluripotent stem cell aggregates in a closed system using a stirring tank bioreactor. Includes). In addition, the methods described herein are suspension of pluripotent stem cells in a closed system without the use of membrane filters and / or centrifugation and / or enzymatic digestion that allow maintenance of sterility in the closed system. And / or enables non-adhesive cultures, reduces costs (eg, for installing centrifuges), and reduces human intervention that helps reduce cross-contamination. Also, the use of membrane filters and / or centrifugation can be included, and the use of enzymatic digestion for the separation of aggregates in further passages of cells or cell aggregates can be included. The use of the methods of the invention in combination with further passages of is conceivable within the scope of the embodiments presented herein.

Previously described methods for the proliferation of pluripotent stem cells in agitated tank bioreactors (eg, Niebruegge et al., Tissue Engineering: Part A, 2008, Volume 14, Issue 10, Pages 1591-1601) It discloses the proliferation of mouse stem cells. Moreover, such previously described methods concentrate stem cell culture for a sufficient period of time, generally maintaining the resulting differentiation of stem cells. In contrast, the methods described herein allow for the proliferation of human stem cells, followed by the separation of aggregates, and the passage of isolated cells, so that the cells pass through proliferation and continuous passage. Retains pluripotency. In addition, the growth and passage methods described herein are performed in a closed system that ensures sterility during the manufacturing process. Generally, the aforementioned stem cell proliferation method uses a Rho-related protein kinase inhibitor (ROCK inhibitor) to maintain cell culture during enzymatic passage. The disadvantage of using ROCK inhibitors for stem cell proliferation and passage is that ROCK inhibitors affect chromosomal stability, presumably causing karyotype abnormalities and genetic drift of stem cells. In contrast, the proliferation and passage of stem cells described herein is performed in the absence of a ROCK inhibitor, thereby reducing karyotype abnormalities and genetic drift, and pluripotency during stem cell passage. Allows the retention of ability.

The methods described herein rely on gravitational sedimentation in combination with automated perfusion in a closed system. Moreover, in some embodiments, the methods described herein include the use of a slicer, thereby allowing enzyme-free separation of cell aggregates between successive passages of cells. .. Certain aforementioned layered gravity sedimentation agents require the cells or cell aggregates to roll on an inclined plane / tube. Disadvantages of such layered gravity sedimentation agents for suspension-aggregation applications are that the cell aggregates have high cell loss due to the efficiency of the growth process, incomplete sedimentation efficiency, and aggregate binding / adhesion to the gravity sedimentation agent. It is susceptible to lowering shear stress. Layered gravity precipitants have been used to separate and discard cell aggregates from cell populations. In contrast, the methods described herein separate and discard single cells (low viability stem cells) while retaining aggregates for subsequent passage.

Other aforementioned methods for medium exchange can temporarily interrupt agitation, thereby allowing the agglomerates to gravitationally settle in the agitation tank bioreactor. After the aggregates have settled, a portion of the used medium can be removed from the bioreactor and replaced with fresh medium. The risk of this approach is that conglomerates of aggregates can occur while the aggregates are concentrated on the bottom surface.

Therefore, this specification describes a method for the proliferation of cell aggregates, including human pluripotent stem cell proliferation, in a stirring tank bioreactor. Continuous or discontinuous agitation of the culture medium provides mixing and aeration, providing a robust environment for cell proliferation. The method uses culture vessels of various sizes to provide ease of operation and protection against cross-contamination. The sensor allows continuous monitoring of dissolved oxygen, pH and media components such as lactic acid and glucose, controls in real time and stores data. Platform software provides the ability to perform continuous or discontinuous perfusion / medium exchange in a closed system.

Usually, during perfusion, there are various methods for keeping cells in culture while removing used medium. One method is to keep the cells in the bioreactor by using capillary fibers or membranes whose pore size is smaller than the size of the cells. Another method is to utilize a "lily pad" floating filter that keeps the cells in the bioreactor while removing the medium. Another method is to use a centrifuge to separate the cells and return them to the bioreactor. Yet another method uses a physical approach, such as acoustics, to capture cells in the cell culture vessel or associated piping while the used medium is being removed.

In contrast, the methods described herein remove used medium without using the filtration system commonly used to keep cells in a bioreactor while allowing removal of medium at the same time. Relies on gravitational sedimentation of cell aggregates to enable. The advantage of this method is the loss of single cells and the maintenance of aggregates, which are largely non-viable in pluripotent stem cell cultures, thereby increasing the overall quality and viability of the culture. ..

By continuously removing used medium and replacing it with new medium, nutrient levels are maintained for optimal growth conditions and cell waste is removed to avoid toxicity. The potential for contamination is reduced when perfusion is performed in a closed system using the methods described herein. Advantageously, the closed systems and cell culture methods described herein utilize gravity sedimentation of cell aggregates, thereby allowing membraneless perfusion, loss due to cell attachment to the filtration membrane and / or It makes it possible to reduce cell damage due to shearing during the filtration process.

The methods described herein are preferably used in closed systems to minimize the risk of culture contamination and cross-cell contamination and to ensure high viability and high cell density are reached. NS. The methods described herein are designed with ease of use and reliability in mind.

Provided herein are methods of describing the proliferation of pluripotent stem cells as suspension aggregates in a stirring tank system with continuous passage. Also, the use of a stirring tank reactor in combination with a slicer for passage is advantageous for pluripotent stem cell proliferation and continuous passage in an integrated closed system, especially for time, drug and labor savings. Examples are also provided herein.

Also provided herein is a specific assembly of tubing connected to a culture vessel (eg, a stirring tank bioreactor) that interacts with a computer controlled peristaltic pump to drive automated media exchange. In one embodiment, the pipe is T-shaped and has a lower vertical pipe, a branch point, and two additional length pipes connected at the branch point. Additional piping is placed on the peristaltic pump controlled by the software. A slow collection rate is used to remove the medium. Due to the vertical nature of the piping, the agglomerates gravitationally settle at a rate that exceeds the flow velocity of the removed medium. The net effect is that during the media removal step, the aggregates remain in optimal cell culture conditions within the vessel (eg, agitated tank bioreactor). The removal of the medium can be continued for up to, for example, 8 hours, 4 hours, etc., but the removal of the medium may be carried out over a much shorter or longer time. After the medium removal step, fresh medium is rapidly added to the container (eg, agitated tank bioreactor) over a period of seconds to minutes or any suitable time. Repeat the medium removal / rapid medium addition cycle for cell culture of the desired length. In another example, perfusion may be discontinuous, and such embodiments are also contemplated within the scope of the embodiments presented herein.

The automated perfusion design described herein is inherently low cost and can be tuned to be perfectly compatible with culture vessels, including agitated tank reactors, and with other culture vessels. No need for filters that increase costs, increase the risk of fouling, and reduce performance. Moreover, the automated perfusions described herein do not include moving parts or electronics that increase complexity, cost, and risk of failure.

The methods provided herein for the growth of cell aggregates in a closed system are:
Steps to provide a cell culture vessel and
Steps to form aggregates in the container,
The step of automatically perfusing cell aggregates in the container,
The step of gravity sedimentation of cell aggregates during perfusion,
Steps to collect and subculture aggregates in a closed system,
including.

In some embodiments, passage in the closed system is in the presence of a Rho-associated protein kinase (ROCK) inhibitor (eg, the concentration of the ROCK inhibitor in the cell culture medium in the vessel is about 10 micromoles). It is done in. In another embodiment of the above method, passage in a closed system is carried out in the substantially absence of an agent that maintains the viability of the passaged monodisperse or disaggregating pluripotent cells. In some such embodiments, the agent that maintains the viability of pluripotent cells is a Rho-associated protein kinase (ROCK) inhibitor. In some embodiments of the above method, passage in a closed system is performed in the absence of a ROCK inhibitor. In some of such embodiments, the ROCK inhibitor is Y27632. As used herein, in one embodiment, "substantially in the absence of an agent that maintains viability of passaged monodisperse or disaggregating pluripotent cells" is passaged. It means that no drug is added to the cell culture medium to maintain the viability of monodisperse or deagglomerating pluripotent cells. In another embodiment, "substantially in the absence of a drug that maintains viability of passaged monodisperse or disaggregating pluripotent cells" is the concentration of the drug in the cell culture medium present in the vessel. Means less than 1 micromol, less than 5 micromoles, or less than 10 micromoles.

In some embodiments, the proliferated cell aggregates are of plant, animal, insect or microbial origin. In some embodiments, the proliferated cell aggregate comprises a human pluripotent stem cell or a differentiated human cell or a combination thereof. In some embodiments, the proliferated cell aggregate comprises human pluripotent stem cells.

In some embodiments, the cell culture vessel is a stirring tank bioreactor. In some embodiments, the automatic perfusion step is performed in a closed system without human intervention.

In some embodiments, the gravity settling chamber is at least 1 cm long and is oriented in a direction that causes gravity settling of cell aggregates.

In some embodiments, automated perfusion is performed at a rate that allows gravitational sedimentation of cell aggregates with a diameter of about 100 microns to about 800 microns. In some embodiments, the above method is continuous. Substitution further comprises the step of further growing one or more of the cell aggregates and / or their progeny. In some of such embodiments, one or more further proliferation of cell aggregates and / or their progeny is carried out by passage to a second vessel with a membrane filter.

In some embodiments, the method comprises one or more additional growths of cell aggregates and / or their progeny, with one or more additional passages taking place in the same container. In other embodiments, the method comprises further proliferation of one or more cell aggregates and / or progeny thereof, with one or more additional passages in a second vessel, in a culture vessel. This is done in the absence of a membrane filter (eg, by gravitational sedimentation of cell aggregates). In another example, the method described above comprises further proliferation of one or more cell aggregates and / or progeny thereof, wherein one or more additional passages have a membrane filter (eg, a floating membrane filter). It is done in the container of 2.

In such embodiments, further proliferation and / or passage can be performed in any order. As an example, the first passage is performed in a culture vessel under membraneless conditions (eg, by gravity sedimentation of cell aggregates), followed by one or more additional growths in the culture vessel with a membrane filter. It is done in. As another example, one or more initial growths and / or passages are performed in a culture vessel containing a membrane filter followed by membraneless filtration conditions (eg, by gravity sedimentation of cell aggregates). Proliferation and / or passage can be performed. Thus, any sequence of growth and / or passage involving membrane filtration or membrane-free filtration is intended within the embodiments described herein, and said sequence is a condition without this membrane. Includes at least one proliferation underneath (eg, due to gravitational sedimentation of cell aggregates).

In some embodiments, continuous passage of cell aggregates is possible by enzyme-free passage with a slicer grid in a closed system. In some of such embodiments, cell aggregates are separated on a slicer grid with blades separated by a distance of about 20 to about 500 microns. In some other embodiments, cell aggregates are separated on a slicer grid with blades separated by a distance of about 100 microns. In some of such embodiments, the cell aggregates are separated by a slicer grid along the tubing and a device for mixing the cell aggregates. In other words, in some embodiments, the cell aggregates are mixed in, for example, the conical bag shown in FIG. 8 prior to separation with a slicer, which is typically combined with the culture vessel in a closed system. Placed between the slicer.

The concentration of cell aggregates used to pass through the slicer affects the recovery of sliced cell aggregates with high viability. Unexpectedly, slices with a cell aggregate concentration of less than about 3 x 10 ^ 6 cells / mL have a cell concentration of more than about 3 x 10 ^ 6 cells / mL using the slicer shape shown in FIGS. 5-7. It was found to produce higher viability samples with higher recovery. Those skilled in the art will recognize that alternative slicer shapes modify threshold concentrations that result in relatively high viability and recovery. Contamination was minimized by maintaining a uniform suspension of aggregates in the flow stream, such as by using the mixer shown in FIG. 8, resulting in higher cell viability and recovery. .. Advantageously, the use of slicers eliminates the need for a Y27632 ROCK inhibitor during the growth of sliced pluripotent stem cell aggregates, because it maintains the viability of a single pluripotent cell. It is different from enzymatically passaged pluripotent stem cell aggregates, which generally require agents such as Y27632 ROCK inhibitors.

In a group of embodiments, the slicer is coated with a hydrophobic material. In some embodiments, the slicer comprises a hydrophobic material.

Certain other embodiments are also contemplated, in which continuous passage of cell aggregates is possible by the separation of cell aggregates in a closed system vessel in the presence of an enzyme. In such embodiments, further proliferation or passage can be performed in the presence of a ROCK inhibitor.

In some embodiments of the methods described above, the average diameter of each proliferated cell aggregate during growth and / or passage is about 800 microns or less in size. In some embodiments of the methods described above, the average diameter of each proliferated cell aggregate during growth and / or passage is about 500 microns or less in size. In some embodiments of the methods described above, the average diameter of each proliferated cell aggregate during growth and / or passage is about 400 microns or less in size. In some embodiments of the methods described above, the average diameter of each proliferated cell aggregate during growth and / or passage is about 300 microns or less in size.

In some embodiments, the volume of the culture vessel is from about 50 mL to about 100 L. In some embodiments, the volume of the culture vessel is from about 50 mL to about 50 L. In some embodiments, the volume of the culture vessel is from about 100 mL to about 10 L. In some embodiments, the volume of the culture vessel is from about 100 mL to about 5 L. In some embodiments, the volume of the culture vessel is from about 150 mL to about 1 L. In some embodiments, the volume of the culture vessel is from about 50 mL to about 20 L. In some embodiments, the volume of the culture vessel is from about 200 mL to about 2 L, or greater than 2 L.

Further provided herein is a method of substituting cell aggregates, in which the size of the cell aggregates is reduced by a slicer grid associated with the bioreactor in a closed system. In a group of embodiments of any of the methods described herein, cell aggregates are passaged in volumes greater than 100 mL. In other words, passage is generally done with a smaller amount of medium and a smaller number of cells. In contrast, the methods of the invention allow the use of large amounts of medium in closed systems, thereby allowing passage of cell aggregates on a bioreactor and / or industrial scale. For example, the use of slicer grids in combination with bioreactors in closed systems for passage of large amounts of cell aggregates, such as greater than 100 mL, has not been disclosed in the art prior to this disclosure. In another embodiment, cell aggregates are passaged in volumes greater than 250 mL, 500 mL, 1 L, 2 L or 5 L. In one embodiment, the slicer grid is a polygonal slicer grid. In one example, the passage of cell aggregates in volumes greater than 100 mL is performed without the addition of a ROCK inhibitor (eg, Y27632) to the medium.

In some embodiments of the method for subculturing cells described above, cell aggregates are separated on a slicer grid with blades separated by a distance of about 20 to about 500 microns. In some embodiments of the method for subculturing cells described above, cell aggregates are separated on a slicer grid with blades separated by a distance of about 100 microns. In some of such embodiments, the cell aggregates are separated by a slicer grid along the tubing and a device for mixing the cell aggregates. In some cases, the slicer is coated with a hydrophobic material. In another example, the slicer comprises a hydrophobic material.

In some embodiments of the methods for subculturing cells described above, the average diameter of each cell aggregate before passage is about 800 microns or less in size. In some embodiments of the methods for subculturing cells described above, the average diameter of each cell aggregate before passage is about 500 microns or less in size. In some embodiments of the methods for subculturing cells described above, the average diameter of each cell aggregate before passage is about 400 microns or less in size. In some embodiments of the methods for subculturing cells described above, the average diameter of each cell aggregate before passage is about 300 microns or less in size.

In another aspect, provided herein is a method for the growth of cell aggregates in a closed system.
With the steps of providing a stirring tank bioreactor vessel,
Steps to form aggregates in the container,
The step of automatically perfusing cell aggregates in the container,
The step of gravity sedimentation of cell aggregates during perfusion,
Steps for collecting cell aggregates and performing enzyme-free slicing,
Includes the step of substituting in a closed system.

In some embodiments of the methods described above, passage in a closed system is performed in the presence of a ROCK inhibitor (eg, the concentration of the ROCK inhibitor in the cell culture medium in the vessel is about 10 micromoles. ). In another embodiment of the above method, passage in a closed system is carried out in the substantially absence of an agent that maintains the viability of the passaged monodisperse or disaggregating pluripotent cells. In some such embodiments, the agent that maintains the viability of pluripotent cells is a Rho-associated protein kinase (ROCK) inhibitor. In some embodiments of the above method, passage in a closed system is performed in the absence of a ROCK inhibitor. In some of such embodiments, the ROCK inhibitor is Y27632. As used herein, in one embodiment, "substantially in the absence of an agent that maintains viability of passaged monodisperse or disaggregating pluripotent cells" is passaged. It means that no drug is added to the cell culture medium to maintain the viability of monodisperse or deagglomerating pluripotent cells. In another embodiment, "substantially in the absence of a drug that maintains viability of passaged monodisperse or disaggregating pluripotent cells" is the concentration of the drug in the cell culture medium present in the vessel. Means less than 1 micromol, less than 5 micromoles, or less than 10 micromoles.

Thus, the methods provided herein allow for several workflows, which include, but are not limited to, (1) enzymatically separated aggregates (eg, Accutase ™), cryopreserved. Aggregation formation in a bioreactor containing stock and / or mechanically sliced aggregates (eg, polygon slicer grid), (2) Propagation method in agitating system: non-perfusion with piping assembly for gravity sedimentation Bioreactor and / or perfusion bioreactor, (3) Continuous passage: Enzymes added to aggregates in a cell culture vessel (eg, Accutase ™ in a bioreactor), aggregates on the outside of the cell culture vessel and / Or contains enzymes added to mechanical passages using a polygonal slicer grid.

Further, the methods described herein include, but are not limited to, agitated suspension bioreactors, locking motion bioreactors, spinner flasks, orbital motion bioreactors, rotary motion bioreactors, and tangential fluid flow bioreactors. Suitable for use in various bioreactors used for suspension culture of. The methods described herein include unfiltered gravity sedimentation for perfusion and enzyme-free passage with a slicer and are compatible with all such bioreactor systems and in such bioreactors. Allows the formation, perfusion, proliferation, collection and passage of closed system aggregates.

Within the scope of the embodiments provided herein, the methods described herein generate cell aggregates grown for research use in order to generate a bank of cells. To be used for therapeutic and / or diagnostic tests (eg, drug testing, toxicology or quality control assays in clinical trials) and / or for the treatment of patients. Administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one cell and / or cell aggregate obtained from the methods described herein to a subject in need thereof. Methods are provided herein.

Also herein is a method of treating a disorder in a subject in need of treatment by administering a therapeutically effective amount of cells and / or aggregates produced by the above method to a subject in need thereof. Provided. The method treats disorders in a subject in need of treatment by administering a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and cells and / or aggregates produced by the method described above. Further includes methods of treatment. It will be appreciated that the methods described herein are also applicable to pluripotent stem cells and differentiated cells.

In some embodiments, the purity and / or homogeneity of the grown cells obtained by the methods described herein and / or for administration to a subject is about 100% (substantially homogeneous). Is. In other embodiments, the purity and / or homogeneity of the grown cells obtained by the methods described herein and / or for administration to a subject is 95% to 100%. In some embodiments, the purity and / or homogeneity of the grown cells obtained by the methods described herein and / or for administration to a subject is 85% to 95%. In the case of a mixture with other cells, the proportions are about 10% -15%, 15% -20%, 20% -25%, 25% -30%, 30% -35%, 35% -40%, It may be 40% to 45%, 45% to 50%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95%.

The choice of formulation for administering cells for a given application depends on a variety of factors. Notable of these are the species of the subject, the nature of the condition being treated, its condition and distribution in the subject, the nature of the other therapies and drugs administered, the optimal route for administration, the route. Survivability, dosing regimens, and other factors apparent to those skilled in the art. For example, the choice of suitable carrier and other additives depends on the exact route of administration and the nature of the particular dosage form. The final formulation of an aqueous suspension of cells / medium usually makes the ionic strength of the suspension isotonic (ie, about 0.1-0.2) and physiological pH (ie, about pH 6.8-). Includes adjusting to 7.5). The final formulation also usually contains a fluid lubricant.

In some embodiments, cells are formulated in injectable form in unit doses such as solutions, suspensions, or emulsions. Suitable pharmaceutical formulations for cell injection are usually sterile aqueous solutions and dispersions. Carriers for injectable formulations are solvents containing, for example, water, saline, phosphate buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. Alternatively, it may be a dispersed medium. One of ordinary skill in the art can readily determine the amount of cells and any additives, vehicles, and / or carriers in the composition administered in the methods of the invention.

The composition will take into account factors such as the age, gender, weight, and condition of the particular patient, and the formulation to be administered (eg, solid vs. liquid), and doses and doses well known to those skilled in the art of medicine and veterinary medicine. It can be administered by technique.

It should be understood that a single dose can be delivered at once, in divided doses, or continuously over a period of time. The entire dose may be delivered to a single location or may be distributed in multiple locations.

In various embodiments, cells may be administered at an initial dose and then maintained by further administration. Cells may be administered first by one method and then by the same method or one or more different methods. Levels can be maintained by ongoing administration of cells. Various embodiments administer the cells first, maintain their levels in the subject, or both by intravenous injection. In various embodiments, other dosage forms are used depending on the patient's condition and other factors discussed elsewhere herein. Appropriate regimens for initial and additional doses, or for sequential doses, may all be the same or varied. Appropriate regimens can be identified by those skilled in the art from this disclosure, the literature cited herein, and knowledge of the art. The dose, frequency and duration of treatment will depend on many factors, including the nature of the disease, the subject, and other therapies that can be administered at the same time. In any of the embodiments described herein, the cells may or may not be differentiated. In any of the embodiments described herein, the cells may be isolated or aggregated. In any of the embodiments described herein, cells and / or cell aggregates can include combinations of pluripotent stem cells and their progeny.

Examples Materials and Methods Materials: Aggregates were cultivated in the GE prototype agitation tank reactors shown in FIGS. 3-4. A Cyclone Labtainer bag was used to hold fresh medium for supply and used medium was collected. Accutase ™ was purchased from MP Biomedical (CA, USA). The mTeSRTM1 medium is STEMCELL ™ Technology Inc. Purchased from (Vancouver, BC, Canada). Y-27632 (ROCK inhibitor) was purchased from Sigma Aldrich (St. Louis, MO). CT2 hESC was obtained from Ren-He Xu at the University of Connecticut Health Center. The gravitational settling piping assemblies used in polygon grid slicers and passages are specially manufactured for this application and were not purchased through commercial vendors.

METHODS: Human embryonic stem cells were adapted from Matrigel ™ to suspension aggregates over 5 passages prior to the stirring tank reactor experiment. Stock cells were confirmed to be karyotyped. Continuous passage was performed using Accutase ™ and the aggregates were reduced to small clusters and single cells that modified the suspended aggregates after seeding. Cell count and viability were measured using Nucleocounter® NC-200 ™ (Chemometec, Denmark).

Human embryonic stem cell line CT2 was a adapted suspension from a feeder-free cell stock and was passaged at least 5 times on a small scale prior to bioreactor culture. Enzymatic passage with Accutase ™ produced populations of single cells and small (<5 cell) clusters. Aggregates were formed 2-12 hours after the addition of single cells / small aggregates to the vessel. The cells formed aggregates with a diameter of about 50-200 μm. It was normal to obtain agglomerate diameter distributions 50 μm above and below the average agglomerate diameter. Most of the agglomerates were within their size, but in some cases some larger agglomerates of about 200-400 μm were formed. Conditions that favor smaller aggregates are preferred because larger aggregates can limit the availability of nutrients, while smaller aggregates result in greater relative growth in culture.

Preferred conditions provide spherical agglomerates that minimize agglomeration. It is important to balance the level of agitation in the bioreactor, as too much agitation leads to shearing, including deformation of the agglomerates, producing an excessive number of non-aggregated single cells. Too little agitation leads to agglomeration of agglomerates.

Not all pluripotent cell lines prefer the same culture conditions. The following parameters have been used for PSC growth, but one of ordinary skill in the art will recognize that other conditions provide PSC growth in similar vessels. That is, the temperature is 37 ° C., the CO ₂ level is 5%, the ambient O ₂ (about 21%) or the reduced O ₂ level, and the stirring speed is continuous or discontinuous at 40 rpm.

Piping assembly for gravity sedimentation and medium replacement in non-perfused bags:
The conceptual configuration of the piping assembly on the stirring tank bioreactor is shown in FIGS. 1 to 4. Assemblies provide, but are not limited to, the following features: (1) Removal of cell / cell culture medium mixture, (2) Separation of cell aggregates from outgoing cell culture medium, (3) Addition of cell culture medium, and (4) Removal of cell culture medium.

Removal of cell aggregate / cell culture medium mixture is achieved by the use of immersion tubes. The immersion tube must be of sufficient length / orientation so that it can be removed from the bioreactor while the cells / medium are installed and operating. Separation of cell aggregates from the outgoing medium is achieved by introducing a gravity settling chamber with sufficient length (height) and diameter to ensure sufficient gravity settings during media removal. .. The design of this chamber is not limited to large diameter tubes, but can also integrate winding pathways if necessary for sufficient cell aggregate separation. One or more piping assemblies operating in parallel can be associated with the bioreactor to increase the rate of media removal / addition. The piping for the addition / removal of fluid must be long enough to ensure a connection to the medium / waste container. Fluid removal is achieved by leaving the fluid addition pathway closed and withdrawing the medium through the fluid removal pathway. Fluid addition is achieved by injecting fresh medium through the fluid addition pathway, leaving the fluid removal pathway closed. The conceptual design of FIGS. 1-4 shows a discontinuous perfusion method with alternating medium removal and medium addition. Another embodiment of this concept is a continuous perfusion method in which separate tubing is used for filter-free media removal and media addition.

In the case of discontinuous perfusion, software control coordinates the removal of certain amounts of used medium and the addition of fresh medium. This method is usually independent of the weight of the container. Preferably, after a predetermined amount of used medium has been removed, an amount of fresh medium is bolus-added according to a predetermined supply schedule. For example, the feed schedule can be set to remove 50 mL of used medium every 2 hours, followed by the addition of 50 mL of fresh medium.

Unfiltered perfusion from agitated tank bioreactor. Approximately 600,000 cells / mL (approximately 97% viability) of CT2 human embryonic stem cells were cultured as suspension aggregates in 250 mL in a stirring tank bioreactor of the GE prototype and stirred at approximately 40 rpm. In other experiments, the suspended aggregates were stirred at 40-75 rpm. The size of the agglomerates in the bioreactor ranged from about 100 μm to about 300 μm (FIG. 12). The gravity settling chamber was tilted at an angle of about 45 degrees and operated along the inner edge of the bioreactor. The inner diameter of the pipe was 3/32 ". The design of the unfiltered perfusion assembly was shown as in FIGS. 3 and 4, allowing both used medium removal and fresh medium addition. In the same pipe. Addition of fresh medium returned any aggregate retained in the tubing to the main bioreactor chamber, exchanging approximately 150 mL / day with a flow rate of 0.2 mL / min to 0.4 mL / min. , Medium was removed from the bioreactor and 8 mL / min was added for fresh medium addition. Medium exchange was not continuous and instead 30 mL was exchanged 5 times daily. 13 million cells at the end of culture. Only (mainly single cells) were found in the waste with a viability of 45%, indicating that little cells in the waste were lost.

Slicer design:
The slicer can be composed of various biocompatible materials. The material must be suitable for sterilization and must have mechanical strength capable of withstanding the stress applied during the flow of the cell sample. The two materials tested for subculture of pluripotent stem cell aggregates were nickel alloys and silicon. Those skilled in the art will recognize that other materials have properties that allow the desired slicer performance for the passage of aggregates. The slicer is designed with a polygonal grid pattern, such as a square or hexagonal grid, with spacing between the walls of the grid of 50 to 400 microns. In some experiments, the slicer was coated with a hydrophobic material to reduce shear and fouling. In the subculture experiments of pluripotent stem cell aggregates below, square and hexagonal grids at 100 μm intervals were used. The slicer was mounted along a pipe that allowed a sterilized flow of aggregates across the slicer through the pipe in a closed system. The slicer may be integrated into the closed system by various fastening mechanisms including, but not limited to, adhesives, molten polymer streams, or tightening. A preferred method is to maintain the agglomerates in suspension via a circulation loop driven by a pump and an in-line conical bag (FIG. 8). The piping leading to the slicer is connected to the main circulation loop, and some of the cell aggregates in the circulation loop are delivered to the slicer via a second pump that operates at a slower rate than the pump that controls the circulation loop. .. Sliced aggregates may be collected in separate containers or reintroduced into the same container.

Slicer performance during cell aggregate passage:
The aggregates passed through the slicer in a flow stream consisting of a volume of 100 mL to 1 L. The advantage of slicers over enzymatic passages is a reduction in time, labor and reagents. Good slicing to a size of about 100 μm was achieved by passing the slicer once or multiple times in a unidirectional or bidirectional flow. The flow rate was controlled to minimize shear. Pluripotent stem cell aggregates that passed through the slicer at a flow rate of 15-150 mL / min demonstrated aggregate slicing performance for size reduction, maintenance of cell viability and subsequent proliferation. Those skilled in the art will recognize that good performance can be achieved at other flow rates. The concentration of cell aggregates used to pass through the slicer was found to affect the recovery of sliced cell aggregates with high viability. Slicer stains can reduce cell recovery and viability. Slices with cell aggregate concentrations below about 3 x 10 ^ 6 cells / mL were found to produce higher viability samples with higher recovery than cell concentrations above about 3 x 10 ^ 6 cells / mL. Contamination was minimized by maintaining a uniform suspension of aggregates in the flow stream, such as by using the mixer shown in FIG. 8, resulting in higher cell viability and recovery. .. A sample image of the sliced aggregate is shown in FIG. Aggregate morphology after slicing includes irregular shapes, rectangular parallelepiped shapes and spherical shapes. With a 100 μm slicer, at least one dimension of the aggregate is reduced to a diameter of about 100 μm. Sliced aggregates were cultured in mTeSR1 and optionally 1-10 μM Y27632 ROCK inhibitor to allow growth. Sliced pluripotency, unlike enzymatically passaged pluripotent stem cell aggregates, which generally require agents such as Y27632 ROCK inhibitors to maintain the viability of single pluripotent cells No Y27632 ROCK inhibitor was required for the growth of embryonic stem cell aggregates. The morphology of the sliced aggregates rapidly reshaped into spheres under culture conditions. The growth rate of sliced aggregates was similar to that of enzymatically passaged cells (FIGS. 10 and 11). Aggregates can be passaged by a slicer without the PBS wash required for enzymatic passage, so slicing passage may take less time than enzymatic passage and will also result in overall effort. few. As will be appreciated by those skilled in the art, slicer functionality and performance will be independent and, but not limited to, other locking, spin or orbital motion platforms or stirring tank bioreactors, including, but not limited to, the Xuri Cellbag bioreactor platform. Compatible with other types of bioreactors.

Although only certain features of the invention have been exemplified and described herein, many modifications and modifications will be made to those skilled in the art. Therefore, it should be understood that the appended claims are intended to include all such modifications and modifications as being within the scope of the true spirit of the invention.

Claims (15)

A method for the growth of cell aggregates in a closed system,
The closed system includes a cell culture vessel, a tubing assembly, a device for mixing cell aggregates, and a slicer grid.
The step of supplying cells to the cell culture vessel and
The step of proliferating the cells in the cell culture vessel to generate the cell aggregate in the cell culture vessel, and
With the step of performing automated perfusion of the cell aggregates in the cell culture vessel
A step of maintaining the cell aggregate in the cell culture vessel by gravity sedimentation of the cell aggregate during the perfusion.
The step of collecting the cell aggregates and
With the step of performing continuous passage of the collected cell aggregates in the closed system
Including
The step of continuous passage includes a step of separating the collected cell aggregates using a device for mixing the slicer grid and the cell aggregates attached along the piping of the piping assembly. , How. The method of claim 1, wherein the step of performing the passage in the closed system is performed in the presence of a Rho-related protein kinase (ROCK) inhibitor. The method of claim 1, wherein the step of performing the passage in the closed system is performed in the substantially absence of an agent that maintains the viability of the passaged monodisperse or disaggregating pluripotent cells. .. The method of claim 3, wherein the agent that maintains said viability of pluripotent cells is a Rho-related protein kinase (ROCK) inhibitor. The method according to one or more of claims 1 to 4, wherein the proliferated cell aggregate comprises human pluripotent stem cells. The method according to one or more of claims 1 to 5, wherein the cell culture vessel is a stirring tank bioreactor. The method of one or more of claims 1-6, wherein the automatic perfusion step is performed in the closed system without human intervention. The method according to one or more of claims 1 to 7, wherein the gravity settling chamber is at least 1 cm in length and is arranged in a direction that causes gravity settling of cell aggregates. The method of one or more of claims 1-8, wherein the automatic perfusion step is performed at a rate that allows gravitational sedimentation of cell aggregates with a diameter of about 100 microns to about 800 microns. The method of one or more of claims 1-9, wherein the cell aggregates are separated by a slicer grid having blades separated by a distance of about 20 to about 500 microns, such as a distance of about 100 microns. The method according to one or more of claims 1 to 10, wherein the slicer is coated with a hydrophobic material or contains a hydrophobic material. The average diameter of each cell aggregates grown is not larger than about 800 microns, for example, the following size about 500 microns, Method according to one or more of claims 1 to 1 1. The volume of the culture vessel is about 50mL~ about 100L, Method according to one or more of claims 1 to 1 2. A method for the growth of cell aggregates in a closed system,
The closed system includes a stirring tank bioreactor vessel, piping assembly, equipment for mixing cell aggregates, and a slicer grid.
The step of supplying cells to the stirring tank bioreactor container,
The step of proliferating the cells in the stirring tank bioreactor container to generate the cell aggregate in the stirring tank bioreactor container, and
With the step of performing automated perfusion of the cell aggregates in the stirring tank bioreactor vessel
A step of maintaining the cell aggregates in the stirring tank bioreactor vessel by gravity sedimentation of the cell aggregates during the perfusion.
The step of collecting the cell aggregates and
With the step of performing continuous passage of the collected cell aggregates in the closed system
Including
The step of continuous passage includes a step of separating the collected cell aggregates using a device for mixing the slicer grid and the cell aggregates attached along the piping of the piping assembly. , How. Performing said passage in said closed system is performed in the substantial absence of an agent that maintains the viability of monodisperse or disaggregating pluripotent cells passaged, according to claim 1 4 Method.

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