Automated Medium Exchange Strategy for Suspension Cells

Abstract

The present invention relates to a method for changing the culture medium of a suspension culture, the suspension culture comprising cells suspended in a culture medium, the method comprising the following steps: (i) transferring a portion of the suspension culture into a container, the container having at least one opening in the bottom; (ii) allowing the cells contained in the portion of the suspension to settle by gravity into at least one opening in the bottom of the container, thereby forming a supernatant; (iii) aliquoting the cells that have settled at the bottom of the container and returning them to the suspension culture; and (iv) discarding the supernatant. TIFF2023505407000003.tif68147

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from European Patent Application No. 19215091.0, filed December 11, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Technical field of the invention The present invention relates to a method for exchanging the culture medium of a suspension culture, wherein the suspension culture comprises cells suspended in the culture medium. [Background technology]

[0003] background Induced pluripotent stem cells (iPSCs) and other adherent cells are traditionally cultured and expanded in static cell culture vessels ("2D culture"). Due to their adherent properties, this type of culture allows for easy medium changes; spent medium is removed, while adherent cells remain on the surface of the cell culture vessel. However, 2D culture can only be scaled and automated to a limited extent, resulting in a large, manual, and cost-intensive workload for, for example, generating large numbers of clinical therapy cells and commercial therapeutic cell products under GMP conditions.

[0004] With regard to commercial bioprocess development, an attractive alternative to traditional 2D cultures is the cultivation of suspension cultures in stirred bioreactors. For example, culturing iPSCs in suspension in the form of iPSC aggregates ("3D culture") has been described several times in the literature (Olmer et al. 2012; Kwok et al. 2018; Amit et al. 2010). 3D suspension cultures offer the possibility of developing efficient, reproducible, and scalable bioprocesses in which culture parameters such as pH, dissolved oxygen (DO), and temperature can be controlled.

[0005] In contrast to traditional 2D culture, medium exchange in suspension cultures remains challenging because cells / cell aggregates must be separated from the medium and the spent medium must be removed while retaining the cells / cell aggregates. In bioprocessing, this can be solved classically by perfusion. Another possibility, already mentioned, is to remove the medium and add fresh medium after discontinuing the stirring and settling of the cells / cell aggregates (see, e.g., Kwok et al. 2018). However, interrupting stirring for extended periods can lead to the formation of even larger aggregates (aggregate fusion). This leads to the formation of larger, more heterogeneous aggregates, which in turn creates differences in mass transfer and signal gradients, leading to spontaneous differentiation and a decrease in the pluripotency of the cell aggregates (see also Lipsitz et al. 2018, Example 1).

[0006] US2016 / 0215257 discloses a method for expanding and passaging cell aggregates containing stem cells and / or differentiated cells, including the use of a sealed system in a stirred tank bioreactor. WO2013 / 109520 discloses a perfusion bioreactor system with a cell aggregate trap. US2017 / 0191022 discloses the use of an acoustic filter to retain cells.

[0007] Thus, there remains a need for a method for exchanging the culture medium of a suspension culture, particularly a method for exchanging the culture medium of a suspension culture, in which the entire process can be performed without removing the cells or aggregates from the system and without exposing the cells to the stresses associated with sedimentation and / or centrifugation for extended periods. The present invention aims to address this need. Summary of the Invention

[0008] This need is solved by the subject matter defined in the claims. Presented herein is a method for exchanging the culture medium of a suspension culture, wherein the suspension culture comprises cells suspended in the culture medium.

[0009] Accordingly, the present invention provides a method for exchanging the culture medium of a suspension culture, comprising the steps of: A suspension culture comprises cells suspended in a culture medium; below: (i) transferring a portion of the suspension culture into a container, the container having at least one opening on its bottom; (ii) allowing the cells in a portion of the suspension to settle by gravity into at least one opening in the bottom of the container, thereby forming a supernatant; (iii) A step of separating the cells that have settled to the bottom of the vessel and transferring them back into the suspension culture; (iv) Discarding the supernatant This relates to a method, including:

[0010] The method may be carried out in a bioreactor, which may be a stirred bioreactor (STR), a rocking motion bioreactor (RM), and / or a multi-parallel bioreactor.

[0011] The suspension culture may be continuously stirred.

[0012] The cells may be essentially uniformly dispersed in the culture medium.

[0013] The container may be tubular. The container may have a conical base. The container may be a pipette tip, a (disposable) bag, or a cone / conical part of a (disposable) bag.

[0014] In step (i), a portion of the suspension culture may be aspirated into a container.

[0015] The cell may be a eukaryotic cell, a fungal cell, such as a yeast cell, e.g., P. pastoris, an insect cell, e.g., Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cell, a bacterial cell, e.g., E. coli, Streptomyces and Salmonella typhimurium cell, or a plant cell. Preferably, the cell is a eukaryotic cell.

[0016] The cells may be adherent cells cultured in suspension.

[0017] The cells may be selected from the group consisting of primary cells, cells obtained from tissues or organs, immortalized cells, and pluripotent stem cells. Preferably, the cells are pluripotent stem cells, preferably induced pluripotent stem cells (iPSCs) or iPSC-derived cells. The cells may be selected from the list consisting of TC-1133, Gibco's human episomal iPSC line, ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027, ATCC ACS-1030, HEK293, HEK293T, BHK21, CHO, NS0, and Sp2 / 0-Ag14. The cells may be human or non-human.

[0018] The cells may be cell aggregates. The cell aggregates may have an average diameter of about 50 to 800 μm, about 150 to 800 μm, at least about 800 μm, at least about 600 μm, at least about 500 μm, at least about 400 μm, at least about 300 μm, at least about 200 μm, at least about 150 μm, about 300 to 500 μm, about 150 to 300 μm, about 50 to 150 μm, about 80 to 100 μm, about 180 to 250 μm, or about 200 to 250 μm.

[0019] The method may further comprise the step of (v) adding a volume equal to the supernatant to the suspension culture.

[0020] In step (ii) of the method of the invention, the period for sedimenting the cells may be at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, at least 16 minutes, at least 17 minutes, at least 18 minutes, at least 19 minutes, or at least 20 minutes.

[0021] At least 70%, at least 80%, at least 90%, at least 95%, at least 97.5%, at least 99%, or essentially all of the cells transferred in step (i) may be sorted (returned) to the suspension culture in step (iii).

[0022] The cells may be grown on microcarrier particles. The suspension culture may be a microcarrier culture.

[0023] In step (ii) of the method of the present invention, a portion of the suspension may be kept static, thereby allowing the cells contained in the portion of the suspension to settle by gravity into at least one opening in the bottom of the container.

[0024] In step (i) of the method of the present invention, a portion of the suspension may be transferred to the container through at least one opening in the bottom of the container.

[0025] In step (iii) of the method of the present invention, the cells that have settled to the bottom of the vessel are sorted through at least one opening in the bottom of the vessel and placed (returned) into the suspension culture. [Brief explanation of the drawings]

[0026] The invention will be better understood in connection with the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[0027] [Figure 1]An exemplary embodiment of the method of the present invention, sometimes referred to as "tip sedimentation" or "pipette tip sedimentation," is shown. A portion (2) of a suspension culture is transferred from the suspension culture to a vessel (1). This can be an automated process, in which the tip of the vessel (1), which has at least one opening (3), is submerged in the suspension culture, and the portion (2) of the suspension culture is aspirated. The exemplary vessel (1), shown in this illustration as a conical pipette tip in cross section, has one opening (3) at its bottom. The vessel, which is a pipette tip in this illustration, is then held stationary for a defined period of time, such as about 3 or about 5 minutes, allowing the cells (22) to settle by gravity in the at least one opening at the bottom of the vessel. Sedimentation can occur while the vessel is submerged in the suspension culture, or the vessel can be removed from the suspension culture again. This creates an essentially cell-free culture medium supernatant (21). After allowing a sufficient number of cells or cell aggregates to settle, the cells (22) are aliquoted and returned to the suspension culture, while the culture medium supernatant (21) is retained. The remaining supernatant (21) may be discarded, for example, by aliquoting it into a waste container. The process shown in Figure 1 may be repeated continuously, allowing for continuous exchange of the culture medium. This continuous exchange can be considered to mimic a perfusion medium exchange process. [Figure 2] The morphology of iPSC aggregates is shown. Images of wells of a 24-well plate containing aggregates harvested from ambr15 suspension cultures are shown. Images were acquired using a Cellavista Cell Imager. Scale bar: 3 mm. [Figure 3] iPSC expansion rate after 4 days is shown. [Figure 4] Figure 1 shows the expression of pluripotency-associated genes. The expression of pluripotency-associated genes was analyzed by flow cytometry on day 4 of iPSC suspension culture. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description of the Invention The present invention is described in detail below and is further illustrated in the accompanying examples and figures.

[0029] Automated medium exchange of suspension cultures in bioreactors remains a challenging task. Manual medium exchange typically involves removing at least a portion of the suspension culture from the bioreactor, e.g., by centrifugation of the cells. This mechanical stress can adversely affect cell viability or function, such as unwanted differentiation of stem cells (see also Lipsitz et al. 2018, Example 1). The medium exchange method of the present invention avoids cell transfer from the bioreactor and stressful mechanical stress, thereby limiting the number of manual interactions and the risk of contamination.

[0030] One possibility for automated medium exchange ("vessel settling") of suspension cultures in bioreactors is to stop agitation and allow the cells to settle to the bottom of the bioreactor. The supernatant can then be aspirated and replaced with fresh medium. However, this also results in mechanical stimulation of the cells, which can lead to unregulated growth and loss of pluripotency (see, e.g., Example 1, Figures 2 and 4). This problem is overcome by the method of the present invention.

[0031] The problems outlined above are solved by the method of the present invention ("chip sedimentation"). Cells in suspension culture in bioreactors are preferably agitated. This is especially important for normally adherent cells, which can form cell aggregates in suspension culture. In current technology, agitation is stopped and the cells or cell aggregates are allowed to settle. After settling, the supernatant is replaced and agitation is started again (see, e.g., Kwok et al. 2018). However, settling has a negative effect on the cells (Lipsitz et al. 2018, Example 1, Figures 2 and 4). Therefore, in the method of the present invention, the bioreactor is operated continuously, preferably without agitation, and stopping agitation is preferably avoided. However, the method of the present invention can also be performed in bioreactors in which the culture medium is not agitated.

[0032] Surprisingly, despite producing well-formed / shaped cell aggregates ( FIG. 2 ) and a dramatically higher percentage of pluripotency marker-positive iPSCs ( FIG. 4 ) compared to the vessel sedimentation method, the method of the present invention ("chip sedimentation") also results in a faster iPSC proliferation rate, as shown in Example 1 and FIG. 4. In other words, the method of the present invention is an improved way of exchanging culture medium in suspension cultures.

[0033] In an exemplary embodiment of the method of the present invention (see also FIG. 1 or Example 1), a portion of the cell culture medium is transferred to a container, e.g., a pipette tip. After transfer, the cells are allowed to settle by gravity at the bottom of the container through at least one opening in the bottom of the container. This causes the cells to collect at the bottom of the portion of the culture medium. That is, a supernatant, preferably essentially cell-free, is formed. After settling, the cells are sorted and returned to the culture medium of the bioreactor, e.g., by ejecting the cells while leaving the supernatant in the container. The supernatant can be discarded. To maintain a constant volume of culture medium in the bioreactor, the discarded supernatant may be replaced with an equal amount of fresh culture medium. Importantly, only a (relatively small) portion of the cells is harvested from the suspension culture, whereas the remaining cells are continuously stirred. Thus, only a small number of cells are present in the container, thereby reducing the time required for medium exchange and, therefore, the time for cell sedimentation during medium exchange. Most importantly, there is no need to remove the cells from the bioreactor.

[0034] Accordingly, the present invention provides a method for exchanging the culture medium of a suspension culture, comprising the steps of: A suspension culture comprises cells suspended in a culture medium; below: (i) transferring a portion of the suspension culture into a container, the container having at least one opening on its bottom; (ii) allowing the cells in a portion of the suspension to settle by gravity into at least one opening in the bottom of the container, thereby forming a supernatant; (iii) A step of separating the cells that have settled to the bottom of the vessel and transferring them back into the suspension culture; (iv) Discarding the supernatant This relates to a method, including:

[0035] When carrying out the method of the present invention, the cells contained in the part of the suspension can be considered to be kept static in step (ii), which means that there is no liquid inflow in step (ii), but the cells only settle by gravity. Thus, step (ii) of the method of the present invention also includes: (ii) maintaining the portion of the suspension static, thereby allowing the cells contained in the portion of the suspension to settle by gravity into at least one opening in the bottom of the container, thereby forming a supernatant. It may also include. Therefore, the present invention also provides a method for exchanging the culture medium of a suspension culture, comprising the steps of: A suspension culture comprises cells suspended in a culture medium; below: (i) transferring a portion of the suspension culture into a container, the container having at least one opening on its bottom; (ii) holding the portion of the suspension static, thereby allowing the cells contained in the portion of the suspension to settle by gravity into at least one opening in the bottom of the container, thereby forming a supernatant; (iii) A step of separating the cells that have settled to the bottom of the vessel and transferring them back into the suspension culture; (iv) Discarding the supernatant The present invention also relates to a method, including:

[0036] A further feature of the present invention may be considered that a portion of the suspension culture is transferred to the vessel and / or cells that have settled to the bottom of the vessel are separated from the vessel through at least one opening in the bottom of the vessel. Thus, the same opening may be used to transfer a portion of the cell suspension to the vessel and to transfer (back) cells that have settled to the bottom of the vessel to the suspension culture. Thus, step (i) of the method of the present invention also comprises: (i) transferring a portion of the suspension culture to a container, the container having at least one opening on the bottom surface, and the portion of the suspension culture being transferred to the container through the at least one opening on the bottom surface of the container; It may also include. Step (iii) also comprises: (iii) dispensing the cells that have settled to the bottom of the vessel through at least one opening in the bottom of the vessel back into the suspension culture. It may also include.

[0037] Therefore, the present invention also provides a method for exchanging the culture medium of a suspension culture, comprising the steps of: A suspension culture comprises cells suspended in a culture medium; below: (i) transferring a portion of the suspension culture into a container, the container having at least one opening on the bottom surface, and the portion of the suspension culture being transferred into the container through the at least one opening on the bottom surface of the container; (ii) allowing the cells in a portion of the suspension to settle by gravity into at least one opening in the bottom of the container, thereby forming a supernatant; (iii) A step of separating the cells that have settled to the bottom of the vessel and transferring them back into the suspension culture; (iv) Discarding the supernatant The present invention also relates to a method, including:

[0038] Therefore, the present invention also provides a method for exchanging the culture medium of a suspension culture, comprising the steps of: A suspension culture comprises cells suspended in a culture medium; below: (i) transferring a portion of the suspension culture into a container, the container having at least one opening on its bottom; (ii) allowing the cells in a portion of the suspension to settle by gravity into at least one opening in the bottom of the container, thereby forming a supernatant; (iii) dispensing the cells that have settled to the bottom of the vessel back into the suspension culture through at least one opening in the bottom of the vessel; (iv) Discarding the supernatant The present invention also relates to a method, including:

[0039] Therefore, the present invention also provides a method for exchanging the culture medium of a suspension culture, comprising the steps of: A suspension culture comprises cells suspended in a culture medium; below: (i) transferring a portion of the suspension culture into a container, the container having at least one opening on the bottom surface, and the portion of the suspension culture being transferred into the container through the at least one opening on the bottom surface of the container; (ii) holding the portion of the suspension static, thereby allowing the cells contained in the portion of the suspension to settle by gravity into at least one opening in the bottom of the container, thereby forming a supernatant; (iii) dispensing the cells that have settled to the bottom of the vessel back into the suspension culture through at least one opening in the bottom of the vessel; (iv) Discarding the supernatant The present invention also relates to a method, including:

[0040] The term "dispensing" as used herein is not limited to applying a force from inside the container. Alternatively, removing cells from outside the container, for example, by sucking the cells from the container, is also contemplated by the present invention. During this process, the supernatant may remain in the container and can then be discarded.

[0041] The amount or volume of discarded culture medium may be replaced by adding a volume equal to the supernatant to the suspension culture. This allows the volume of the suspension culture to be kept constant. Therefore, the method of the present invention may further include step (v): adding a volume equal to the supernatant to the suspension culture. The method of the present invention may also be used to increase or decrease the volume of a suspension culture. For example, the (total) volume of a suspension culture is increased by adding a volume to the suspension culture that exceeds the volume of the supernatant. Therefore, the method of the present invention may further include step (v): adding a volume to the suspension culture that exceeds the volume of the supernatant. Conversely, the (total) volume of a suspension culture is decreased by adding a volume to the suspension culture that is less than the volume of the supernatant. Therefore, the method of the present invention may further include step (v): adding a volume to the suspension culture that is less than the volume of the supernatant.

[0042] As used herein, the term "suspension culture" refers to a type of cell culture in which single cells or small cell aggregates are able to function and grow, preferably in an agitated growth medium, thus forming a suspension (see definition in Chemistry: "small, solid particles suspended in a liquid"). This is in contrast to adherent culture, in which cells are attached to a cell culture vessel, which may be coated with extracellular matrix (ECM) proteins. In suspension culture, preferably, ECM proteins are not added to the cells and / or culture medium. Suspension cultures are preferably essentially free of solid particles, such as beads, microspheres, microcarrier particles, etc. Cells or cell aggregates are not solid particles in this context. In one embodiment, the cells are not in a microcarrier (suspension) culture.

[0043] The methods of the present invention can also be used for medium exchange in microcarrier cultures, i.e., for cells grown on microcarrier particles. Thus, the cells may be grown on microcarrier particles. As used herein, "microcarrier particles" refers to a support matrix that allows adherent cell growth in a bioreactor. Microcarriers are typically 125-250 μm spherical, and their density allows cells to be maintained in suspension with gentle agitation. Microcarriers can be made from a number of different materials, including DEAE-dextran, glass, polystyrene plastic, acrylamide, collagen, and alginate. Surface chemistries can include extracellular matrix proteins, recombinant proteins, peptides, and positively or negatively charged molecules. Several types of microcarriers are commercially available, including alginate-based (GEM, Global Cell Solutions), dextran-based (Cytodex, GE Healthcare), collagen-based (Cultispher, Percell), and polystyrene-based (SoloHill Engineering) microcarriers. These microcarriers may differ in porosity, specific gravity, optical properties, presence of animal components, and surface chemistry.

[0044] As used herein, a "growth medium" or "culture medium" is a liquid designed to support the growth of microorganisms, cells, or small plants. Different types of media are used to grow different types of cells. One skilled in the art can determine which culture medium is best for a particular cell type.

[0045] The methods of the present invention are preferably carried out in a bioreactor. As used herein, the terms "reactor" and "bioreactor" are used interchangeably and refer to a closed culture vessel configured to provide a dynamic fluid environment for cell culture. Bioreactors may be stirred and / or agitated. Examples of stirred reactors include, but are not limited to, stirred tank bioreactors, wave-mixed / rocking bioreactors, top-bottom stirred bioreactors (i.e., stirred reactors with piston movement), spinner flasks, shake flasks, shaker bioreactors, paddle mixers, and vertical wheel bioreactors. Stirred reactors may be configured to accommodate cell culture volumes ranging from approximately 2 mL to 20,000 L. Preferred bioreactor volumes may be up to 50 L. An exemplary bioreactor suitable for the methods of the present invention is the ambr15 bioreactor available from Sartorius Stedim Biotech. Bioreactors may be stainless steel or disposable bioreactors. A bioreactor may consist of a single vessel or may comprise several bioreactors in parallel. Disposable bioreactors may be made of glass or plastic. Disposable bioreactors may be stirred tank bioreactors or rocking motion bioreactors. Examples: Sartorius STR, RM, ambr15, ambr250. The pH of the culture medium may be controlled by the bioreactor, preferably by CO2 supply, and may be kept in the range of 6.6 to 7.6, preferably around 7.4.

[0046] The bioreactor may be a stirred bioreactor (STR). STRs are available, for example, from Sartorius Stedim Biotech, and include, but are not limited to, the BIOSTAT® A / B / B-DCU / Cplus / D-DCU, ambr® 15, and ambr® 250. The bioreactor may be a rocking motion bioreactor (RM). RMs are available, for example, from Sartorius Stedim Biotech, and include, but are not limited to, the BIOSTAT® RM and the BIOSTAT® RM TX. The bioreactor may be a multi-parallel bioreactor, for example, from Sartorius Stedim Biotech, and include, but are not limited to, the ambr® 15 and ambr® 250.

[0047] In some embodiments, the volume of the culture vessel in the bioreactor is about 50 mL to about 20,000 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 50 mL to about 2,000 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 50 mL to about 200 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 50 mL to about 100 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 50 mL to about 50 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 50 mL to about 20 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 50 mL to about 10 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 50 mL to about 1 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 100 mL to about 10 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 100 mL to about 5 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 150 mL to about 1 L. In some embodiments, the volume of the culture vessel in the bioreactor is about 1 L to about 1,000 L.

[0048] As outlined herein, the cells in suspension culture are preferably dispersed in the culture medium without settling.Therefore, the suspension culture is preferably agitated.Continuous agitation can essentially uniformly disperse the cells in the culture medium / suspension culture, and can help stem cells such as iPSCs maintain pluripotency (see also Example 1 and Figure 4).Therefore, the cells are preferably essentially uniformly dispersed in the culture medium.

[0049] The term "vessel" as used herein relates to any vessel suitable for holding a suspension culture comprising cells suspended in a culture medium. The vessel may be tubular, i.e., may have a cylindrical shape. One base of the cylinder is preferably essentially oriented toward gravity. The vessel or cylinder may have a conical base rather than a flat base. At least one opening may be located at the very bottom of the conical surface. Such a vessel with a conical base may also be referred to as a pipette tip. Therefore, any type of pipette tip is contemplated as the vessel of the present invention.

[0050] However, the container is not limited to a pipette tip or the like. The container may also be a bag. As used herein, a "bag" refers to a flexible container, preferably a flexible container made of plastic, comprising at least one flexible tube. The flexible tube may be submerged in the suspension culture. That is, when the container is a bag, the "bottom" of the container may refer to the end of the tube submerged in the suspension culture. The tube may have a conical / conical end, which may be submerged in the suspension culture and corresponds to the bottom of the container described herein. Examples of bags include, but are not limited to, disposable bags commercially available from Sartorius Stedim biotech, which may be sold as Flexsafe® 2D or Flexsafe® 3D bags. The bag may also be a disposable bag.

[0051] To allow the sedimented cells to be removed from the vessel and returned to the suspension culture, the vessel has at least one opening at the bottom through which the cells can be removed. A portion of the cells may be transferred to the vessel through the at least one opening at the bottom. However, the present invention also contemplates the use of a different opening to transfer a portion of the suspension culture to the vessel. When a (closed) bioreactor is used, the vessel is preferably inside the bioreactor.

[0052] The portion of the suspension culture can be transferred by any suitable means. For example, the transfer is controlled by aspiration, for example by applying a slight negative pressure that aspirates the portion into the container. Thus, in step (i), a portion of the suspension culture may be aspirated into the container.

[0053] The method of the present invention can generally be used for any cells that can be cultured in cell culture, i.e., for adherent cell cultures. Advantageously, the method is used to replace the culture medium of a suspension culture, in which case it is most important to separate the cells from the culture medium. In this context, "suspended in a culture medium" refers to cells cultured in suspension, regardless of whether they are actually suspension cells. Therefore, the method of the present invention can also be used for adherent cells, provided that the adherent cells are suspended in a culture medium. Thus, the cells may be adherent cells cultured in suspension.

[0054] Adherent cells cultured in suspension, i.e., unable to adhere to the culture vessel, may form cell aggregates. As used herein, the terms "aggregate" and "cell aggregate" may be used interchangeably and refer to a plurality of cells, e.g., (induced) pluripotent stem cells. In this case, the binding between cells is caused by cell-cell interaction (e.g., by biological adhesion to each other). The biological adhesion may be, for example, due to surface proteins, such as integrins, immunoglobulins, cadherins, selectins, or other cell adhesion molecules. For example, cells may spontaneously combine in suspension to form cell-cell adhesion (e.g., self-assembly), thereby forming aggregates. In some embodiments, cell aggregates may be substantially homogeneous (i.e., may generally contain the same type of cells). In other embodiments, cell aggregates may be heterogeneous (i.e., may contain multiple types of cells).

[0055] The methods of the present invention are suitable for cell aggregates. The cell aggregates may vary in size. The cell aggregates may have an average diameter of about 50-800 μm, about 150-800 μm, at least about 800 μm, at least about 600 μm, at least about 500 μm, at least about 400 μm, at least about 300 μm, at least about 200 μm, at least about 150 μm, about 300-500 μm, about 150-300 μm, about 50-150 μm, about 80-100 μm, about 180-250 μm, or about 200-250 μm.

[0056] The cells may be any cells that can be cultured in suspension, e.g., prokaryotic or eukaryotic cells, with eukaryotic cells being preferred in one embodiment. The cells may be selected from the group consisting of primary cells, cells obtained from tissues or organs, immortalized cells, and pluripotent stem cells. Preferably, the cells are pluripotent stem cells, more preferably induced pluripotent stem cells (iPSCs), or cells derived from iPSCs. "Cells derived from iPSCs" refer to differentiated cells or cells that have differentiated into a specific cell type that can no longer differentiate into any cell type in the body. Methods for differentiating different cell types from iPSCs are known to those skilled in the art. "Cells derived from iPSCs" may relate to cardiac cells and / or tissue, liver cells and / or tissue, kidney cells and / or tissue, brain cells and / or tissue, pancreatic cells and / or tissue, lung cells and / or tissue, skeletal muscle cells and / or tissue, gastrointestinal cells and / or tissue, neuronal cells and / or tissue, skin cells and / or tissue, bone cells and / or tissue, bone marrow, adipocytes and / or tissue, connective cells and / or tissue, retinal cells and / or tissue, vascular cells and / or tissue, stromal cells, or cardiomyocytes. Methods for producing cardiac tissue are known from WO2015 / 025030 and WO2015 / 040142. The cells may also be differentiated into, for example, cardiomyocytes or stromal cells in a bioreactor or outside a bioreactor. These differentiated cells may also be cultured in a bioreactor using the methods of the present invention. Cells obtained from tissues or organs may be obtained from cardiac cells and / or tissue, liver cells and / or tissue, kidney cells and / or tissue, brain cells and / or tissue, pancreatic cells and / or tissue, lung cells and / or tissue, skeletal muscle cells and / or tissue, gastrointestinal cells and / or tissue, neuronal cells and / or tissue, skin cells and / or tissue, bone cells and / or tissue, bone marrow, adipocytes and / or tissue, connective cells and / or tissue, retinal cells and / or tissue, vascular cells and / or tissue, stromal cells or cardiac muscle cells.

[0057] The cell may be a mammalian cell, such as a human, dog, mouse, rat, pig, or ape, such as a cynomolgus monkey, to name a few. Preferably, the cell is a human cell. Other preferred cells include fungal cells, such as yeast cells, e.g., P. pastoris, insect cells, e.g., Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells, bacterial cells, e.g., E. coli, Streptomyces, and Salmonella typhimurium cells, or plant cells.

[0058] As used herein, the term "pluripotent stem cells" (PSCs) refers to cells that can differentiate into any cell type in the body. Thus, pluripotent stem cells offer a unique opportunity to differentiate into essentially any tissue or organ. Currently, the most widely utilized pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Human ESC lines were first established by Thomson and colleagues (Thomson et al. (1998), Science 282:1145-1147). Human ESC research has recently enabled the development of a new technology for reprogramming somatic cells into ES-like cells. Yamanaka and colleagues pioneered this technology in 2006 (Takahashi & Yamanaka (2006), Cell, 126:663-676). The resulting induced pluripotent cells (iPSCs) behave very similarly to ESCs and, importantly, can also differentiate into any cell type in the body. Therefore, in one embodiment, the term iPSC includes ESC. However, in the context of the present invention, these pluripotent stem cells are preferably not produced using a process that involves the modification of human germline genetic identity or the use of human embryos for industrial or commercial purposes. Preferably, the pluripotent stem cells are derived from primates, more preferably from humans.

[0059] Suitable artificial PSCs can be obtained, for example, from the NIH human embryonic stem cell registry, the European Bank of Induced Pluripotent Stem Cells (EBiSC), the Stem Cell Repository of the German Center for Cardiovascular Research (DZHK), or ATCC, to name a few. Induced pluripotent stem cells are also available for commercial use, for example, from the NINDS Human Sequence and Cell Repository (https: / / stemcells.nindsgenetics.org), which is operated by the US National Institute of Neurological Disorders and Stroke (NINDS) and distributes human cell resources widely to academic and industrial researchers. An exemplary cell line that can be used in the present invention is cell line TC-1133, an induced (unedited) pluripotent stem cell derived from umbilical cord blood stem cells. This cell line can be obtained, for example, directly from NINDS, USA. Preferably, TC-1133 is GMP-compliant. Further exemplary iPSC cell lines that can be used in the present invention include, but are not limited to, Gibco™ human episomal iPSC line (Order No. A18945, Thermo Fisher Scientific), or iPSC cell lines available from ATCC: ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027, or ATCC ACS-1030.Alternatively, those skilled in the art of reprogramming can easily generate suitable iPSC lines using known protocols, such as those described in Okita et al., "A more efficient method to generate integration-free human iPS cells," Nature Methods, Vol. 8, No. 5, May 2011, pp. 409-411, or Lu et al., "A defined xeno-free and feeder-free culture system for the derivation, expansion, and direct differentiation of transgene-free patient-specific induced pluripotent stem cells," Biomaterials 35 (2014) 2816e2826.

[0060] The cells may be selected from the group consisting of TC-1133, Gibco's human episomal iPSC line, ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027, ATCC ACS-1030, HEK293, HEK293T, BHK 21, CHO, NS0, Sp2 / 0-Ag14.

[0061] As described herein, the (induced) pluripotent stem cells used in the present invention can be obtained from any suitable cell type (e.g., stem cells, such as mesenchymal stem cells or epithelial stem cells, or differentiated cells, such as fibroblasts) and from any suitable source (body fluid or tissue). Examples of such sources (body fluids or tissues) include umbilical cord blood, skin, gums, urine, blood, bone marrow, any compartment of the umbilical cord (e.g., the amniotic membrane or Wharton's jelly of the umbilical cord), the umbilical-placental junction, placenta, or adipose tissue, to name just a few. One example is the isolation of CD34-positive cells from umbilical cord blood by magnetic cell separation using an antibody specifically directed against CD34, followed by reprogramming, as described in Chou et al. (2011), Cell Research, 21:518-529. Baghbaderani et al. (2015), Stem Cell Reports, 5(4):647-659, show that the iPSC generation process could comply with good manufacturing practice regulations to generate the cell line ND50039.

[0062] Therefore, the pluripotent stem cells preferably meet the requirements of good manufacturing practice.

[0063] The method of the present invention outlined herein requires the sedimentation of suspended cells in step (ii). The time required for sedimentation may vary with the size, shape, and mass of the cells. It is within the knowledge of those skilled in the art to determine the time required for sufficient cell sedimentation. Thus, in step (ii) of the method of the present invention, the period for sedimenting cells may be at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, at least 16 minutes, at least 17 minutes, at least 18 minutes, at least 19 minutes, or at least 20 minutes.

[0064] By "sufficient sedimentation" is meant that at least 70%, at least 80%, at least 90%, at least 95%, at least 97.5%, at least 99%, or essentially all of the cells transferred in step (i) are sorted (returned) to suspension culture in step (iii). Thus, at least 70%, at least 80%, at least 90%, at least 95%, at least 97.5%, at least 99%, or essentially all of the cells transferred in step (i) are preferably sorted (returned) to suspension culture in step (iii).

[0065] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents, and reference to "the reagent" includes reference to equivalent steps and methods known to those skilled in the art that may be modified or used in place of the methods described herein.

[0066] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0067] The term "and / or" whenever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0068] The term "less than" or, similarly, the term "greater than" does not include a specific number thereof.

[0069] For example, "less than 20" means less than the indicated number. Similarly, "greater than" or "greater than" means greater than or exceeding the indicated number. For example, "greater than 80%" means greater than or exceeding 80% of the indicated number.

[0070] Throughout the following specification and claims, unless otherwise required by context, the word "comprise" and inflections such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" may be substituted with the terms "containing" or "including," and sometimes, when used herein, may be substituted with the term "having." As used herein, "consisting of" excludes elements, steps, or ingredients not specified.

[0071] The term "including" means "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0072] As used herein, the terms "about," "approximately," or "essentially" mean within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a particular value or range. It also includes the specific number. That is, "about 20" includes the number 20.

[0073] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc., described herein, as such may vary. The methodology used herein is for the purpose of illustrating particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0074] All publications, whether supra or infra, cited throughout the text of this specification (including all patents, patent applications, scientific publications, manuals, etc.) are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that the material incorporated by reference conflicts or is inconsistent with this specification, the present specification supersedes such material.

[0075] The contents of all publications and patent documents cited herein are incorporated by reference in their entirety. [Example]

[0076] A further understanding of the present invention and its advantages will be apparent from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0077] material and method Unless otherwise specified, the following materials and methods were used throughout the examples.

[0078] cell culture ·Cells: TC1133, NINDS ·Seeding density: 5x10 5 cells / mL ·Culture conditions: 37℃, pH7.4, dO 50%, 300rpm downward stirring (downstirr) Start medium change on day 2

[0079] material ambr15 cell culture 24 Disposable Bioreactor, Low Temperature, No Sparger, Part Number: 001-2B81 StemMACS iPS-Brew XF, basal medium, order number: 130-107-086 StemMACS™ iPS-Brew XF 50x Supplement; Order Number: 130-107-087 Ambr15 bioreactor, Sartorius Stedim Nucleocounter NC-200 Type 900-0201 Cellavista Cell Imager Flow cytometer: BD LSR II Special Order System

[0080] Medium exchange Container Sedimentation: 1. Stop the incubation station agitation. 2. Allow the mixture to rest for 5 minutes to allow the aggregates to settle to the bottom. 3. Aspirate the medium by placing the pipette tip 1.4 cm away from the bottom of the container. 4. Add fresh medium 5. Start agitation on the incubation station 6. Total medium change: 66% Chip Sedimentation: 1. Load 900 μl of cell suspension into the pipette tip 2. Leave the suspension in the tip and let it rest for 5 minutes. 3. Return 100 μl to the container. Discard any remaining spent medium. 4. Add 800 μl of fresh medium to the vessel. 5. Repeat steps 1 to 4 six times. 6. Total medium change: 57%

[0081] Example 1: Evaluation of vessel sedimentation and tip sedimentation strategies as a means to exchange medium in iPSC cultures at maximum capacity in the ambr15 system We compared two different medium exchange strategies for culturing iPSCs in the ambr15 at a maximum capacity of 24 vessels. To test both strategies in parallel, the vessel sedimentation strategy was performed in culture station 1, and the tip sedimentation strategy was performed in culture station 2. The maximum capacity of the ambr15 system was mimicked as follows: vessel setup included repeated cycles of activated agitation (860 seconds; simulating a medium exchange in a vessel in the opposite culture station) and inactivated agitation (881 seconds; simulating a vessel exchange in the same culture station). This period was empirically determined in advance. A 2.5-hour pause was included between each tip sedimentation medium exchange.

[0082] result morphology Before starting the medium change, the morphology of the iPSC aggregates was similar (Figure 2). After the medium change, the aggregates in the vessel sedimentation strategy were scattered or tubular-shaped and large, indicating that the aggregates had fused. In contrast, the aggregates in the tip sedimentation strategy were round and small.

[0083] Cell number and expansion rate After 4 days of suspension culture using the vessel sedimentation strategy, cell numbers increased 3.61-fold. During the same period, the tip sedimentation strategy resulted in an average increase of 21.81-fold (Figure 3).

[0084] pluripotency After 4 days of suspension culture using the tip-sedimentation strategy, expression of pluripotency-related genes (OCT4, TRA-1-60, and NANOG) was high (90% OCT4 / TRA-1-60 double-positive cells, 91% OCT4 / NANOG double-positive cells, Figure 4). Expression of cells cultured using the vessel-sedimentation strategy was significantly lower (11% OCT4 / TRA-1-60 double-positive cells, 9% OCT4 / NANOG double-positive cells, Figure 4).

[0085] analysis The tip settling strategy is more suitable than the vessel settling strategy for medium exchange of cell cultures, especially iPSCs, at maximum capacity in bioreactors such as the ambr15 system. Given the insufficient expression of pluripotency-associated markers, the vessel settling strategy may not be suitable for medium exchange at all, especially in the ambr15 system at its maximum capacity of 24 vessels. The reason for this is likely that, without agitation, aggregates would fuse during this period, leading to spontaneous differentiation.

[0086] References TIFF2023505407000002.tif102166

Claims

1. 1. A method for exchanging the culture medium of a suspension culture, comprising: A suspension culture comprises cells suspended in a culture medium; below: (i) transferring a portion of the suspension culture into a container, the container having at least one opening on its bottom; (ii) allowing the cells in a portion of the suspension to settle by gravity into at least one opening in the bottom of the container, thereby forming a supernatant; (iii) A step of separating the cells that have settled at the bottom of the container and returning them to the suspension culture; (iv) Discarding the supernatant A method comprising:

2. The bioreactor is preferably a stirred bioreactor, a rocking motion bioreactor, and / or a multi-parallel bioreactor. The method of claim 1, wherein the method is carried out in

3. The suspension culture is continuously stirred; and / or the cells are essentially uniformly dispersed in the culture medium; 10. The method of any one of the preceding claims.

4. the container is tubular; and / or the container has a conical base; and / or The container is a pipette tip, a (disposable) bag or a cone / cone-shaped part of a (disposable) bag, 10. The method of any one of the preceding claims.

5. 10. The method of claim 1, wherein in step (i) a portion of the suspension culture is aspirated into a container.

6. 10. The method of any one of the preceding claims, wherein the cell is a eukaryotic cell, such as a human cell, a fungal cell, such as a yeast cell, for example, a P. pastoris cell, an insect cell, for example, a Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cell, a bacterial cell, for example, an E. coli, Streptomyces and Salmonella typhimurium cell, or a plant cell.

7. 10. The method of any one of the preceding claims, wherein the cells are adherent cells cultured in suspension.

8. 10. The method of any one of the preceding claims, wherein the cells are selected from the group consisting of primary cells, cells obtained from tissues or organs, immortalized cells, pluripotent stem cells, preferably the cells are pluripotent stem cells, more preferably induced pluripotent stem cells (iPSCs).

9. 2. The method of any one of the preceding claims, wherein the cells are selected from the group consisting of TC-1133, Gibco's human episomal iPSC line, ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027, ATCC ACS-1030, HEK293, HEK293T, BHK 21, CHO, NS0, Sp2 / 0-Ag14.

10. 10. The method of any one of the preceding claims, wherein the cell is a human cell.

11. 10. The method of any one of the preceding claims, wherein the cells are cell aggregates, and the cell aggregates preferably have an average diameter of about 50-800 μm, about 150-800 μm, at least about 800 μm, at least about 600 μm, at least about 500 μm, at least about 400 μm, at least about 300 μm, at least about 200 μm, at least about 150 μm, about 300-500 μm, about 150-300 μm, about 50-150 μm, about 80-100 μm, about 180-250 μm, or about 200-250 μm.

12. (v) adding an equal volume of the supernatant to the suspension culture 10. The method of any one of the preceding claims, further comprising:

13. 10. The method of any one of the preceding claims, wherein in step (ii), the period for sedimenting the cells is at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, at least 16 minutes, at least 17 minutes, at least 18 minutes, at least 19 minutes, or at least 20 minutes.

14. 10. The method of claim 1, wherein at least 70%, at least 80%, at least 90%, at least 95%, at least 97.5%, at least 99%, or essentially all of the cells transferred in step (i) are sorted (returned) into suspension culture in step (iii).

15. 15. The method of any one of claims 1-7, 9-10, and 12-14, wherein the cells are grown on microcarrier particles and / or the suspension culture is a microcarrier culture.

16. 10. The method of claim 9, wherein in step (ii), a portion of the suspension is kept static, thereby allowing the cells contained in the portion of the suspension to settle by gravity into at least one opening in the bottom of the container.

17. 10. The method according to any one of the preceding claims, wherein in step (i) a portion of the suspension is transferred to the container through at least one opening in the bottom of the container.

18. 10. The method of claim 9, wherein in step (iii), the cells that have settled to the bottom of the container are sorted through at least one opening in the bottom of the container and placed (returned) into the suspension culture.

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